Process of making isoprenoid phosphinylformic acid squalene synthetase inhibitors

ABSTRACT

Compounds which are inhibitors of cholesterol biosynthesis (by inhibiting de novo squalene biosynthesis), and thus are useful as hypocholesterolemic agents and antiatherosclerotic agents, are provided which have the structure ##STR1## wherein R 2  is a metal ion, lower alkyl or H; 
     R 3  is a metal ion or lower alkyl; 
     R is R 1  --(CH 2 ) n  --, R 1  --(CH 2 ) m  --O-- or 
     R 1  --(CH 2 ) m  --OCH 2  --, wherein n is 1 to 4 and m is 0 to 3, and 
     R 1  is R 5  --Q 1  --Q 2  --Q 3  --wherein R 5 , Q 1 , Q 2  and Q 3  are as defined herein. 
     New intermediates, new methods of preparation and a method for using such compounds to inhibit cholesterol biosynthesis are also provided.

This is a division of application Ser. No. 650,823, filed Feb. 5, 1991now U.S. Pat. No. 5,107,0, which is a division of application Ser. No.408,974, filed Sept. 18, 1989 now U.S. Pat. No. 5,025,003.

FIELD OF THE INVENTION

The present invention relates to new isoprenoid phosphinylformic acidcompounds which are useful in inhibiting cholesterol biosynthesis byinhibiting de novo squalene production, to hypocholesterolemic andantiatherosclerotic compositions containing such compounds and to amethod of using such compounds for inhibiting cholesterol biosynthesisand atherosclerosis.

BACKGROUND OF THE INVENTION

Squalene synthetase is a microsomal enzyme which catalyzes the reductivedimerization of two molecules of farnesyl pyrophosphate (FPP) in thepresence of nicotinamide adenine dinucleotide phosphate (reduced form)(NADPH) to form squalene (Poulter, C. D.; Rilling, H. C., in"Biosynthesis of Isoprenoid Compounds", Vol. I, Chapter 8, pp. 413-441,J. Wiley and Sons, 1981 and references therein). This enzyme is thefirst committed step of the de novo cholesterol biosynthetic pathway.The selective inhibition of this step should allow the essentialpathways to isopentenyl tRNA, ubiquinone, and dolichol to proceedunimpeded. Squalene synthetase, along with HMG-CoA reductase has beenshown to be down-regulated by receptor mediated LDL uptake (Faust, J.R.; Goldstein, J. L.; Brown, M. S. Proc. Nat. Acad. Sci. USA, 1979, 76,5018-5022), lending credence to the proposal that inhibiting squalenesynthetase will lead to an up-regulation of LDL receptor levels, as hasbeen demonstrated for HMG-CoA reductase, and thus ultimately should beuseful for the treatment and prevention of hypercholesterolemia andatherosclerosis.

One approach to inhibitors of squalene synthetase is to design analogsof the substrate FPP. It is clear from the literature that thepyrophosphate moiety is essential for binding to the enzyme. However,such pyrophosphates are unsuitable as components of pharmacologicalagents due to their chemical and enzymatic lability towards allylic C-0cleavage, as well as their susceptibility to metabolism by phosphatases.

P. Ortiz de Montellano et al in J. Med. Chem., 1977, 20, 243-249describe the preparation of a series of substituted terpenoidpyrophosphate (Table A), and have shown these to be competitiveinhibitors of the squalene synthetase enzyme. These substances retainthe unstable allylic pyrophosphate moiety of FPP.

                  TABLE A                                                         ______________________________________                                         ##STR2##                                                                     No.         X         Y          Z                                            ______________________________________                                        1           CH.sub.3  CH.sub.3   H                                            2           H         H          H                                            3           C.sub.2 H.sub.5                                                                         H          H                                            4           I         H          H                                            5           H         I          H                                            6           CH.sub.3  H          SCH.sub.3                                    ______________________________________                                    

Corey and Volante, J. Am. Chem. Soc. 1976, 98, 1291-3, have prepared FPPanalog A and presqualene pyrophosphate (PSQ-PP) analog B as inhibitorsof squalene biosynthesis. (Presqualene pyrophosphate is an intermediatein the conversion of FPP to squalene). These inhibitors possessmethylene groups in place of the allylic oxygen moiety of FPP andPSQ-PP, but still retain the chemically and enzymatically unstablepyrophosphate linkage. ##STR3##

Poulter and co-workers have prepared cyclopropane C (Sandifer, R. M., etal., J. Am. Chem. Soc. 1982, 104, 7376-8) which in the presence ofinorganic pyrophosphate is an intermediate analog inhibitor of theenzyme squalene synthetase. ##STR4##

Altman and co-workers, Bertolino, A., et al., Biochim. Biophys. Acta.1978, 530, 17-23, reported that farnesyl amine and related derivatives Dinhibit squalene synthetase, but provide evidence that this inhibitionis non-specific and probably related to membrane disruption. ##STR5##

Poulter, C.D., et al, J. Org. Chem., 1986, 51, 4768, prepared compound Ein a demonstration of a synthetic method, but did not report anybiological data. ##STR6##

Poulter, C. D., Stremler, K. E., J.A.C.S., 1987, 109, 5542 describes thesynthesis and biological evaluation of compounds having structure F.These compounds were evaluated as alternative substrates for avian liverand lemon peel farnesyl diphosphate cyclase. ##STR7##

McClard, R. W. and Poulter, C. D., et al., J.A.C.S. 1987, 109, 5544,reported that phosphinylphosphonates G and H were competitive inhibitorsof the 1'-4-condensation between isopentenyl diphosphate and geranyldiphosphate catalyzed by avian liver farnesyl diphosphate synthetase.Phosphinylphosphonates G and H had Ki's of 19μM and 71μM, respectively.They also reported the speculative isolation of the farnesylphosphinylphosphonate I, and the geranyl phosphinylphosphonate J fromthe enzymatic reaction of G with geranyl pyrophosphate or dimethylallylpyrophosphate, respectively. The structures of I and J were tentativelyassigned based on relative TLC mobilities. They hypothesized that Icould be a potential inhibitor of squalene synthetase. ##STR8##

Capson, T. L., PhD dissertation, June 1987, Dept. of MedicinalChemistry, the University of Utah, Abstract, Table of Contents, pp. 16,17, 40-43, 48-51, Summary, and T. L. Capson, C. D. Poulter et al, J.Org. Chem., 1988, 53, 5903-5908 disclose cyclopropanes of the structureK ##STR9## as intermediate analog inhibitors of squalene synthetase.

Biller and coworkers, "Isoprenoid (Phosphinylmethyl)phosphonates asInhibitors of Squalene Synthetase," J. Med. Chem., 1988, 31, 1869,synthesized analogues of FPP, 2a-d and 3a,b, where the allylic andanhydride oxygen atoms are replaced with carbon. The PMP subunit therebyserves as a stable surrogate for the diphosphate. They demonstrate thatisoprenoid (phosphinylmethyl)phosphonates (PMPs) are effectiveinhibitors of squalene synthetase, binding to the enzyme with affinitycomparable to FPP itself. ##STR10##

DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is providedphosphorus-containing isoprenoid phosphinylformic acid compounds whichinhibit cholesterol biosynthesis, and thus are useful ashypocholesterolemic and antiatherosclerotic agents and have thefollowing structure ##STR11## wherein R² is a metal ion, lower alkyl orH;

R³ is a metal ion or lower alkyl;

R is R¹ --(CH₂)--, R¹ --(CH₂)_(m) O-- or

R¹ --(CH₂)_(m) OCH₂ --, wherein n is 1 to 4, m is 0 to 3; and R¹ is R⁵-Q¹ -Q² -Q³ - wherein Q¹, Q² and Q³ are independently: ##STR12## or abond, with the stipulation that if Q¹ is a bond, then Q² and Q³ must bebonds, and if Q² is a bond, then Q³ is a bond; R⁶ is H, lower alkyl,fluoro or fluoroalkyl (e.g. CH₂ F, CF₃); R⁷ is H, fluoro, lower alkyl oralkylthio; R⁸ is H, fluoro, trimethylsilyl or lower alkyl; R⁹ is H, orlower alkyl; ##STR13## R¹⁶ --C.tbd.C-CH₂ --(wherein R¹⁶ is lower alkylor H), or CH₃ (CH₂)_(p) -- where p is 2 to 7; R¹⁰ and R¹¹ areindependently hydrogen, lower alkyl such as methyl or ethyl, fluoro,lower alkenyl or fluoroalkyl or R¹⁰ and R¹¹ can be taken together toform (CH₂)_(s), where s is 2 to 7; R¹² is hydogen, lower alkyl, fluoroor lower alkenyl; R¹³ and R¹⁴ are independently lower alkyl such asmethyl or ethyl; with the proviso that if all of Q¹, Q² and Q³ arebonds, then R¹⁰ and R¹¹ cannot both be H, and R⁵ cannot be CH₃ (CH₂)_(p)--, with p<4.

The formula I compounds of the invention include all stereoisomersthereof.

Unless otherwise indicated, the term "lower alkyl" or "alkyl" asemployed herein alone or as part of another group includes both straightand branched chain hydrocarbons containing 1 to 8 carbons in the normalchain, preferably 1 to 4 carbons, such as methyl, ethyl, propyl,isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl or isohexyl.

The term "lower alkenyl" or "alkenyl" as used herein by itself or aspart of another group refers to straight or branched chain radicals of 2to 12 carbons, preferably 3 to 6 carbons in the normal chain, whichinclude one double bond in the normal chain, and which may include anaryl or alkyl substituent, such as vinyl, 2-propenyl, 2-butenyl,3-phenyl-2-propenyl, 2-pentenyl, 2-hexenyl, 2-heptenyl, 2-octenyl,2-nonenyl, 2-decenyl, 2-undecenyl, 2-dodecenyl and the like.

The term "halogen" or "halo" as used herein refers to chlorine, bromine,fluorine, and iodine.

The term "haloalkyl" as used herein refers to any of the lower alkylgroups defined above substituted with a halogen as defined above, forexample CH₂ F, CF₃ and the like.

The term "metal ion" refers to alkali metal ions such as sodium,potassium or lithium and alkaline earth metal ions such as magnesium andcalcium.

Thus, the compounds of formula I of the invention include the followingtypes of compounds: ##STR14##

Preferred are those compounds of formula I, IA, IB and IC wherein R¹ is##STR15## n is 1, 2 or 3, m is 1 or 2, R² is H or a metal ion, and R³ islower alkyl, a metal ion or H.

The compounds of the invention may be prepared according to thefollowing reaction sequences. ##STR16##

As seen in Reaction Scheme I, compounds of the invention of formula IAmay be prepared starting with the alcohol II wherein R is R¹ --(CH₂)_(n)--, that is

    R.sup.1 --(CH.sub.2).sub.n --OH                            II

which is converted to the corresponding mesylate by treating II withmesyl chloride, organic base such as triethylamine, in an organicsolvent such as methylene chloride, to form the mesylate ##STR17## Theresulting mesylate III is treated with a metal halide such as lithiumbromide in tetrahydrofuran or sodium iodide in acetone while heating toa temperature within the range of from about 45 to about 65° C. to formthe halide IV

    R.sup.1 --(CH.sub.2).sub.n --Hal (Hal=Br or I) .           IV

Halide IV is then subjected to a Grignard reaction, for example, bytreating IV with a stirred suspension of magnesium turnings and1,2-dibromoethane, in the presence of diethyl ether under an inertatmosphere such as argon, to form a Grignard solution referred to inReaction Scheme I as

    R-Metal                                                    A

wherein R is R¹ --(CH₂)_(n) --.

Alternatively, R-Metal (R=lithium) is prepared by treating IV with analkyl lithium such as n-butyl or t-butyllithium or lithium metal, indiethyl ether or tetrahydrofuran at a temperature of from about -78° C.to about 25° C., with a ratio of alkyllithium or lithium metal to IV offrom about 1:1 to about 2.5:1.

The anion solution is reacted with phosphite V

    ClP--(OR.sup.2a).sub.2                                     V

(wherein R^(2a) is lower alkyl) in the presence of an organic solventsuch as diethyl ether or tetrahydrofuran, under an inert atmosphere suchas argon, at a temperature within the range of from about -78° C. toabout 25° C., employing a molar ratio of V:A of within the range of fromabout 1:1 to about 4:1, to form the phosphonite B

    R.sup.1 --(CH.sub.2).sub.n --P(OR.sup.2a).sub.2.           B

Phosphonite B is treated with a chloroformate VII

    ClCO.sub.2 R.sup.3a                                        VII

(wherein R^(3a) is lower alkyl) such as ethyl chloroformate, employing amolar ratio of VII:B of within the range of from about 1:1 to about10:1, to form the diester of the invention ID. Diester ID, in an inertorganic solvent such as methylene chloride, may then be subjected todealkylation by treating with excess bromotrimethylsilane (TMSBr) oriodotrimethylsilane (TMSI) in the presence of 2,4,6-collidine orbis(trimethylsilyl)trifluoroacetamide and then treating with oneequivalent of a strong inorganic base such as aqueous NaOH, KOH, LiOH orMg(OH)2, optionally in the presence of an alcohol such as methylalcohol, to form the salt IE or with excess strong base (greater thantwo equivalents) to form the salt IF. The salt IE may be treated withstrong acid such as HCl to form the free acid IG.

Compounds of the invention of formula IC may be prepared employing theReaction Scheme I except that the starting alcohol II is treated with asolution of base such as potassium hydride or sodium hydride and thenwith an iodide of the structure VIII

    I--CH.sub.2 --Sn(n--C.sub.4 H.sub.9).sub.3                 VIII

employing a molar ratio of VIII:II of within the range of from about 1:1to about 1.5:1, in the presence of an inert organic solvent such astetrahydrofuran, diethylether or dimethyl formamide to form the stannaneof the structure IX

    R.sup.1 --(CH.sub.2).sub.m --O--CH.sub.2 --Sn(n--C.sub.4 H.sub.9).sub.3. IX

The stannane IX is dissolved in an inert organic solvent such as diethylether or tetrahydrofuran, cooled to a temperature within the range offrom about -90° C. to about 0° C., while under an inert atmosphere suchas argon, and treated with a strong base such as n-butyllithium indiethyl ether, tetrahydofuran or other inert organic solvent, under aninert atmosphere such as argon. The resulting solution is then used inplace of the anion solution A in Scheme I, as described above, to formthe phosphite B¹

    R.sup.1 --(CH.sub.2).sub.m --O--CH.sub.2 --P(OR.sup.2a).sub.2, B.sup.1

the diester ID¹ ##STR18## (wherein R^(2a) and R^(3a) are lower alkyl),and the salts IE¹ and IF¹ ##STR19## and the free acid IG¹ ##STR20##

In an alternative synthesis, as seen in Reaction Scheme II, compounds offormula IA or IC of the invention may be prepared by treating A'

    R.sub.a -Metal                                             A'

wherein R_(a) is R¹ --(CH₂)_(n) -- or R¹ --(CH₂)_(m) O--CH₂ -- (preparedas described above) with phosphite V, employing the procedure as set outabove with respect to Scheme I, employing a molar ratio of V:A' ofwithin the range of from about 1:1 to about 5:1.

The reaction mixture is quenced with deaerated water to form thephosphonous ester C ##STR21## (where R^(2a) is lower alkyl) which is anovel intermediate.

The phosphonous ester C may then be treated with a chloroformate VII inthe presence of chlorotrimethylsilane, and an organic base such astriethylamine or diisopropylethylamine to form the diester ID, ID¹. IDmay be converted to IE or IF, and ID¹ may be converted to IE¹ and IF¹according to Scheme I.

The phosphonous ester C may be directly hydrolyzed by treatment with astrong base such as potassium hydroxide, sodium hydroxide or lithiumhydroxide optionally in the presence of an organic solvent such asmethanol, ethanol or tetrahydrofuran, and the resulting salt is treatedwith a strong acid such as dilute hydrochloric acid or dilute sulfuricacid to form phosphonous acid D ##STR22## which is a novel intermediate.

The phosphonous acid D may be treated with a chloroformate VII in thepresence of chlorotrimethylsilane, and an organic base, such astriethylamine or diisopropylethylamine to form the monoester IG, IG¹##STR23## The monoester IG, IG¹ may be converted to the correspondingmonometal salt IE or IE¹ or dimetal salt IF or IF¹ by treating with oneequivalent of base or excess base respectively, as describedhereinbefore with respect to Reaction Scheme I.

Referring to Reaction Scheme III, compounds of the invention of formulaIB may be prepared starting with alcohol ##STR24## which is treated withdichlorophosphinylformate ester F ##STR25## (where R^(3a) is loweralkyl) in the presence of an organic solvent such as tetrahydrofuran,diethyl ether or dichloromethane, under an inert atmosphere such asargon, at a temperature within the range of from about -80 to about 0°C., employing a molar ratio of F:E of within the range of from about 1:1to about 5:1.

Where it is desired to form the monosalt IH, the reaction mixture istreated with one equivalent of base such as sodium hydroxide orpotassium hydroxide. Where the dimetal salt IJ is desired, the reactionmixture is treated with excess base.

The alcohol starting material II or E where m is 1 or n is 1, that is E¹

    R.sup.1 --CH.sub.2 --OH                                    E.sup.1

may be prepared according to the following reaction sequence (followingthe procedure of E. J. Leopold, Organic Synthesis 1985, 64, pp 164-173)##STR26##

The alcohol starting material II or E where m is 2 or n is 2 that is E²

    R.sup.1 --CH.sub.2 CH.sub.2 --OH                           E.sup.2

may be prepared according to the following reaction sequence: ##STR27##

The alcohol starting material II or E where m is 3 or n is 3, that isII³ ##STR28## may be prepared according to the following reactionsequence ##STR29##

The alcohol starting material where n=4, that is ##STR30## may beprepared according to the following reaction sequence ##STR31##

Examples of starting material II or E that is R¹ --(CH₂)_(m) or _(n)--OH and m is 0, 1, 2 or 3 and n is 1, 2, 3 or 4 suitable for use hereininclude the following which are either known in the literature or aresimple derivatives of known compounds prepared by employing conventionalprocedures.

It will be appreciated that the compounds listed in the following tablerepresent all possible stereoisomers.

    ______________________________________                                        R.sup.1(CH.sub.2).sub.m or nOH (m is 0 to 3, n is 1 to 4) where               R.sup.1 is R.sup.5Q.sup.1Q.sup.2Q.sup.3 as follows in A. through              ______________________________________                                        F.                                                                            A.                                                                             ##STR32##                                                                                 R.sup.10       R.sup.11                                          ______________________________________                                        1.           C.sub.2 H.sub.5                                                                              CH.sub.3                                          2.           CH.sub.3       C.sub.2 H.sub.5                                   3.           n-C.sub.3 H.sub.7                                                                            CH.sub.3                                          4.           CH.sub.3       n-C.sub.4 H.sub.9                                 5.           t-C.sub.4 H.sub.9                                                                            CH.sub.3                                          6.           (CH.sub.2).sub.s'                                                             s' = 4 to 6                                                      7.           H              H                                                 8.           F              F                                                 9.           CH.sub.2 F     CH.sub.3                                          10.          CHCH.sub.2     H                                                 ______________________________________                                        B.                                                                                 ##STR33##                                                                           alkyl(CH.sub.2).sub.p                                              ______________________________________                                            1.     CH.sub.3 (CH.sub.2).sub.p where p is 3 to 7                            2.                                                                                    ##STR34##                                                         C.  1.                                                                                    ##STR35##                                                             2.                                                                                    ##STR36##                                                         ______________________________________                                        D.                                                                                 ##STR37##                                                                            R.sup.11   R.sup.6    R.sup.6'                                    ______________________________________                                        1.          C.sub.2 H.sub.5                                                                          C.sub.2 H.sub.5                                                                          CH.sub.3                                    2.          CH.sub.3   CH.sub.3   C.sub.2 H.sub.5                             3.          CH.sub.3   C.sub.2 H.sub.5                                                                          C.sub.2 H.sub.5                             4.          C.sub.2 H.sub.5                                                                          C.sub.2 H.sub.5                                                                          C.sub.2 H.sub.5                             5.          CH.sub.3   C.sub.2 H.sub.5                                                                          CH.sub.3                                    6.          CH.sub.3   H          CH.sub.3                                    7.          CH.sub.3   CH.sub.3   H                                           8.          H          H          H                                           9.          CH.sub.3   CH.sub.3   H                                           10.         H          H          H                                           ______________________________________                                        E.                                                                             ##STR38##                                                                               R.sup.7    R.sup.6   R.sup.8                                       ______________________________________                                        1.         H          F         H                                             2.         H          H         F                                             3.         H          CH.sub.3  CH.sub.3                                      4.         CH.sub.3 S CH.sub.3  H                                             5.         F          CH.sub.3  H                                             6.         CH.sub.3   CH.sub.3  H                                             7.         H          CH.sub.3  CH.sub.3                                      8.         H          CF.sub.3  H                                             9.         H          F         H                                             10.        H          CH.sub.3  (CH.sub.3).sub.3 Si                           11.        H          CH.sub.3  F                                             ______________________________________                                        F.  Other examples of R.sup.1 include the following                           ______________________________________                                         1.                                                                                ##STR39##                                                                 2.                                                                                ##STR40##                                                                 3.                                                                                ##STR41##                                                                 4.                                                                                ##STR42##                                                                 5.                                                                                ##STR43##                                                                 6.                                                                                ##STR44##                                                                 7.                                                                                ##STR45##                                                                 8.                                                                                ##STR46##                                                                 9.                                                                                ##STR47##                                                                10.                                                                                ##STR48##                                                                     ##STR49##                                                                     ##STR50##                                                                     ##STR51##                                                                     ##STR52##                                                                     ##STR53##                                                                     ##STR54##                                                                ______________________________________                                    

The compounds of Formula I of the invention inhibit cholesterolbiosynthesis by inhibition of de novo squalene production. Thesecompounds inhibit the squalene synthetase enzyme and, in addition, someof the compounds of Formula I of the invention inhibit other enzymes inthe pathway from isopentenyl diphosphate to squalene, that is, farnesyldiphosphate synthetase and isopentenyl diphosphate-dimethylallyldiphosphate isomerase.

Thus, the compounds of the invention are useful in treatingatherosclerosis to inhibit progression of disease and in treatinghyperlipidemia to inhibit development of atherosclerosis. In addition,the compounds of the invention may increase plasma high densitylipoprotein cholesterol levels.

As squalene synthetase inhibitors, the compounds of the invention mayalso be useful in inhibiting formation of gallstones and in treatingtumors.

The compounds of the invention may also be employed in combination withan antihyperlipoproteinemic agent such as probucol and/or with one ormore serum cholesterol lowering agents such as Lopid (gemfibrozil), bileacid sequestrants such as cholestyramine, colestipol, polidexide(DEAE-Sephadex) as well as clofibrate, nicotinic acid and itsderivatives, neomycin, p-aminosalicyclic acid, bezafibrate and the likeand/or one or more HMG CoA reductase inhibitors such as lovastatin,pravastatin, velostatin or simvastatin.

The above compounds to be employed in combination with the squalenesynthetase inhibitor of the invention will be used in amounts asindicated in the Physicians' Desk Reference (PDR).

Inhibition of squalene synthetase may be measured by the followingprocedure.

Rat liver microsomal squalene synthetase activity is measured usingfarnesyl diphosphate as substrate and quantitating squalene synthesisusing gas chromatographic analysis. The assay was developed by modifyingconditions originally described by Agnew (Methods in Enzymology 110:357,1985).

Preparation of Rat Liver Microsomes

Livers are dissected from 2 or 3 decapitated Sprague Dawley rats and arequickly transferred to ice cold buffer (potassium phosphate, 0.05M, (pH7.4); MgCl₂, 0.004M; EDTA, 0.001M; and 2-mercaptoethanol 0.01M) andrinsed thoroughly. The livers are minced in cold buffer (2.0 ml/g) andhomogenized using a Potter-Elvejhem homogenizer. The homogenate iscentrifuged at 5,000×g, 10 minutes (4° C.), and the supernatant pouredthrough 2 layers of cheese cloth. The supernatant is then centrifuged at15,000×g for 15 minutes (4°). Again the supernatant is filtered through2 layers of cheese cloth, and centrifuged a third time at 100,000×g for1.0 hour at 4° C. Following centrifugation the microsomal pellet isresuspended in a volume of buffer equivalent to 1/5 the volume of theoriginal homogenate, and homogenized in a ground glass homogenizer.Aliquotted microsomes are frozen at -80° C., and retain activity for atleast two months.

Enzyme Assay

Reaction Mixtures are prepared in 50 ml round bottom pyrex glass tubeswith tight-fitting, teflon-lined, screw caps. Tubes are cooled to 4° C.,and the following components are added in sequence:

    ______________________________________                                        1.    Potassium phosphate buffer                                                                            0.36   ml                                             0.275 M, pH 7.4)                                                        2.    KF (55 mM)              0.36   ml                                       3.    NADPH (5.0 mM, freshly prepared)                                                                      0.36   ml                                       4.    H.sub.2 O (or H.sub.2 O + test compound)                                                              0.16   ml                                       5.    MgCl.sub.2 (27.5 mM)    0.36   ml                                       6.    Microsomal Enzyme (0.48 mg                                                                            0.20   ml                                             microsomal protein in homogeni-                                               zation buffer)                                                                                        1.8    ml                                       ______________________________________                                    

This mixture is equilibrated under N₂ at 4° C. for 5-15 minutes.Reaction mixtures are then warmed to 30° C., and the enzyme reactioninitiated by adding 0.2 ml of farnesyl pyrophosphate (219μM) prepared inH₂ O. Each tube is again overlayered with N₂, and incubated at 30° C.for 60 minutes. The reaction is stopped by the addition of 1.0 ml KOH(40%). Ethanol (95%) (spectral grade) (1.0 ml) is added to each tube.Docosane (5 nmoles in hexane) is added to each tube as an internalstandard. The mixture is saponified at 65° C. for 30 minutes. The tubesare cooled to room temperature and extracted twice with 10.0 ml spectralgrade hexane.

The upper organic phase fractions are pooled in glass 20.0 mlscintillation vials and reduced in volume to ≃1.0 ml under a stream ofN₂. The sample is then transferred to acid-washed, conical bottom, glass(1.0 ml) microvials, and brought to dryness under N₂. The residue isresuspended in 50 μl hexane (spectral grade), and these samples are spunat 1000 rpm at room temperature for 10 minutes. Following centrifugationapproximately 40 μl of supernatant is transferred to 100 μl acid-washedmicrovials with septa/crimp-top caps (compatible with theHewlett-Packard GC auto injector).

Gas Chromatography

Two μL of each sample is injected onto a fused silica megabore DB-17column (15M×0.525 mm) (J&W Scientific) using a splitless mode ofinjection. Gas flow rates are listed below:

    ______________________________________                                        Make up gas (helium)                                                                          20 ml/min.                                                    Air             400 ml/min.                                                   Hydrogen        30 ml/min.                                                    Carrier (helium)                                                                              15 ml/min.                                                    Septum purge vent                                                                              5 ml/min.                                                                    (Septum purge off 0.00                                                        min., on at 0.5 min.)                                         ______________________________________                                    

The injector temperature is 200° C., and the FID detector temperature isset at 270° C. Oven temperature is programmed through a two rampsequence as follows:

    ______________________________________                                        Oven:                                                                         ______________________________________                                        Initial temperature 180° C., initial time 10 minutes                   Ramp one: 20° C./minute                                                Second temperature 250° C., second time 10 minutes                     Ramp two: 20° C./minute                                                Third temperature 260° C., third time 10 minutes                       (Equilibration time 1.0 minute)                                               ______________________________________                                    

Using this gas chromatographic system, docosane (internal standard) hasa retention time of 3.6-3.7 minutes, and squalene has a retention timeof 14.7-14.9 minutes. The amount of squalene in each reaction mixture isdetermined by obtaining the areas under the squalene and docosane peaksand using the following formula to calculate the amount of squalene(nmoles) in the total reaction mixture. ##EQU1##

Compounds Testing

Compounds are dissolved in H₂ O and added to reaction mixtures prior toaddition of farnesyl pyrophosphate substrate. All reaction mixtures arerun in duplicate, at several concentrations. Additionally, all compoundI₅₀ values are derived from composite dose response data.

A further aspect of the present invention is a pharmaceuticalcomposition consisting of at least one of the compounds of Formula I inassociation with a pharmaceutical vehicle or diluent. The pharmaceuticalcompostion can be formulated employing conventional solid or liquidvehicles or diluents and pharmaceutical additives of a type appropriateto the mode of desired administration. The compounds can be administeredto mammalian species including humans, monkeys, dogs, etc. by an oralroute, for example, in the form of tablets, capsules, granules orpowders, or they can be administered by a parenteral route in the formof injectable preparations. The dose for adults is preferably between200 and 2,000 mg per day, which can be administered in a single dose orin the form of individual doses from 1-4 times per day.

A typical capsule for oral administration contains active ingredient(250 mg), lactose (75 mg) and magnesium stearate (15 mg). The mixture ispassed through a 60 mesh sieve and packed into a No. 1 gelatin capsule.

A typical injectible preparation is produced by asceptically placing 250mg of sterile active ingredient into a vial, asceptically freeze-dryingand sealing. For use, the contents of the vial are mixed with 2 ml ofphysiological saline, to produce an injectible preparation.

The following Examples represent preferred embodiments of the presentinvention.

Introduction to Experimental

All temperatures are reported in degrees Centigrade.

¹ H and ¹³ C chemical shifts are reported as δ-values with respect toMe₄ Si (δ=0). ³¹ P spectra were measured on a JEOL FX90Q FT-NMRspectrometer, at 36.2 MHz, utilizing the ¹ H decoupled mode. The ³¹ Pdata were obtained using 85% H₃ PO₄ as an external reference (δ=0).Coupling constants J are reported in Hz. Chemical ionization massspectra (CI-MS) were determined with a Finnigan TSQ-4600 instrumentequipped with a direct exposure probe using the indicated reagent gases.Fast atom bombardment mass spectra (FAB-MS) were recorded on a VGAnalytical ZAB-2F spectrometer. Ions were sputtered (8keV Xe) from amatrix containing dithiothreitol, dithioerythritol, DMSO, glycerol andwater.

All reactions were carried out under an atmosphere of dry argon ornitrogen. The following reagents and solvents were distilled prior touse from the indicated drying agents, where applicable: CH₂ Cl₂,2,4,6-collidine, and diisopropylamine (CaH₂); THF and diethyl ether (K,benzophenone); N,N-diethyltrimethylsilylamine and oxalyl chloride.Benzene was passed through neutral alumina (activity I) and stored over4A-molecular sieves. Lithium bromide was dried at 100° C. over P₂O₅.(E,E)-Farnesol was purchased from Aldrich Chemical Company.

TLC was performed on E. Merck Silica Gel 60 F-254 plates (0.25 mm) or E.Merck Cellulose F plates (0.1 mm). Flash chromatography was carried outusing E. Merck Kieselgel 60 (230-400 mesh).

Reverse-phase chromatographic purification of PMP salts was carried onCHP20P gel (75-150 μ), a highly porous, polystyrene-divinyl benzenecopolymer available from Mitsubishi Chemical Industries. The indicatedgeneral procedure was followed: An FMI Model RP-SY pump was utilized forsolvent delivery. A column of CHP20P (2.5 cm diameter, 12-22 cm height)was slurry packed and washed with water (500-1000 mL), and a basic,aqueous solution of the crude salt was applied to the top of the column.Typically, the column was eluted with water, followed by a gradientcomposed of increasing concentrations of acetonitrile or methanol inwater. The gradient was created by placing the tip of a tightlystoppered separatory funnel containing 300-500 mL of the organicsolvent, or an aqueous-organic mixture, just beneath the surface of areservoir containing 300-500 mL of pure water. To start the gradient,the stopcock of the separatory funnel was opened, so that as the solventwas withdrawn by the pump from the reservoir, it was replaced with thesolvent from the separatory funnel. HPLC-grade solvents and Lectrostillsteam distilled water were employed. Fractions were collected (10-15 mLeach) at a flow rate of 5-10 mL per minute. Those fractions thatcontained pure product as judged by TLC were pooled, the organicsolvents were evaporated and the aqueous residue was lyophilized todryness.

EXAMPLE 1(E,E)-[Ethoxy(5,9,13-trimethyl-4,8,12-tetradecatrienyl)phosphinyl]formicacid, ethyl ester A. Bishomofarnesol (1)(E,E)-3,7,11-Trimethyl-2,6,10-dodecatrienyl bromide

A solution of 1.00 g (4.5 mmol) of (E,E)-farnesol (Aldrich, furtherpurified by flash chromatography) in 10 ml of distilled diethyl ether at0° C. under argon in the dark was treated dropwise with a solution of195 μL (2.05 mmol, 0.45 eq.) of PBr₃ in 2 ml of diethyl ether. Theresultant mixture was stirred at 0° C. for one hour, then quenched withwater and separated. The organic phase was washed with 5 ml of H₂ O, 5ml of saturated NaHCO₃, and 5 ml of brine, dried over Na₂ SO₄ andevaporated to give 1.26 g (98%) of crude bromide as a clear oil.

TLC Silica (2:8 ethyl acetate:hexane) R_(f) =0.69.

¹ H NMR (CDCl₃, 270 MHz): δ 5.52 (t, 1H, J=8.5 Hz), 5.08 (m, 2H), 4.01(d, 2H, J=8.5 Hz), 1.9-2.2 (m, 8H), 1.73 (s, 3H), 1.68 (s, 3H), 1.60 (s,6H) ppm.

(2) (E,E)-5,9,13-Trimethyl-4,8,12-tetradecatrienoic acid,1,1-dimethylethyl ester

To a solution of 9.60 ml (68.5 mmol, 1.5 eq.) of diisopropylamine in 100ml of THF at -78° C. under argon was added 28.2 ml (45.0 mmol, 1.0 eq.)of 1.6M n-butyllithium in hexanes over 20 minutes. After warming to 0°C. for 15 minutes, the solution was recooled to -78° C. and 6.05 ml (45mmol, 1.0 eq) of t-butyl acetate was added over 20 minutes. After anadditional 15 minutes, 16.0 ml (92 mmol, 2.05 eq.) ofhexamethylphosphoramide (HMPA) was added, followed by a solution of12.53 g (45.0 mmol) of Part A(1) farnesyl bromide in 100 ml of THF over20 minutes. The reaction was stirred at -78° C. for 2.5 hours, quenchedwith saturated NH₄ Cl and allowed to warm to room temperature. Afterdiluting with 400 ml of ethyl acetate, the mixture was washed with four100 ml portions of water, and 200 ml of brine, dried over MgSO₄ andevaporated to provide 12.96 g of crude product as a yellow oil.Purification by flash chromatography on 1 kg of silica gel, eluted with1:9 ethyl acetate:petroleum ether afforded 9.39 g (65%) of titlecompound as a pale yellow oil.

TLC Silica gel (2:98 ethyl acetate:hexane) R_(f) =0.16.

IR(neat) 2977, 2925, 2857, 1733, 1452, 1368, 1258, 1149 cm⁻¹.

¹ H NMR(CDCl₃, 270 MHz): δ 5.10 (m, 3H), 2.25 (m, 4H), 1.9-2.1 (m, 8H),1.68 (s, 3H), 1.62 (s, 3H), 1.59 (s, 6H), 1.44 (s, 9H) ppm.

Mass Spec (CI-CH₄ /N₂ O ) (+ ions) m/e 165 (M+H-C₄ H₈), 247, 183, 137,68, 57. (- ions) m/e 319 (M-H), 279, 251, 100.

(3) Bishomofarnesol

To a stirred solution of 5.00 g (15.6 mmol) of Part (2) compound in 45ml of dry diethyl ether at 0° C. under argon was added 592 mg (15.6mmol, 1 mol - eq.) of lithium aluminum hydride, and the resultingsuspension was stirred at room temperature for 20 hours. After coolingto 0° C., the reaction was quenched by treating with 5 ml of H₂ O, 5 mlof 15% NaOH, and 15 ml of H₂ O and stirring the suspension for 1/2 hour.After adding Na₂ SO₄, the slurry was filtered through Celite, washingwell with diethyl ether and evaporated to obtain 3.62 g of crudeproduct. Purification by flash chromatography on 300 g of silica gel,eluted with 1:9 ethyl acetate:petroleum ether provided 3.516 g (90%) ofbishomofarnesol as a colorless liquid.

TLC Silica gel (2:8 ethyl acetate:hexane) R_(f) =0.19

IR(neat) 3330, 2964, 2926, 2873, 2958, 1448, 1384, 1107, 1059, 401 cm⁻¹.

¹ H NMR(CDCl₂, 270 MHz): δ 5.10 (m, 3H), 3.63 (t, 2H, J=6.5 Hz), 1.9-2.2(m, 10H), 1.68 (s, 3H), 1.62 (2, 3H), 1.60 (s, 7H) ppm.

Mass Spec (CI-CH₄ /N₂ O, + ions) m/e 251 (M+H), 249 (M+H-H₂), 137, 123,109, 69.

B. (E,E)-5,9,13-Trimethyl-4,8,12-tetradecatrien-1-ol, methanesulfonateester

To a stirred solution of 2.02 g (8.07 mmol) of bishomofarnesol (preparedas described in Example 1, Part A) in 20 mL of dichloromethane at 0° C.was added 2.2 mL (16.1 mmol) of triethylamine followed by 0.69 mL (8.90mmol) of methanesulfonyl chloride, dropwise over 15 minutes. Afterstirring for 1.5 hours at 0° C., the reaction was diluted withdichloromethane, washed with 20 mL each of 10% HCl, saturated NaHC03 andbrine, dried (MgSO₄) and evaporated to give 2.71 g ("100%") of the crudetitle mesylate as a colorless oil.

TLC Silica gel (CH₂ Cl₂) R_(f) =0.46.

¹ H NMR (CDCl₃, 270 MHz): δ 5.09 (t, 3H, J=6.5 Hz), 4.21 (t, 2H, J=7.0Hz), 2.99 (s, 3H), 1.9-2.2 (m, 10H), 1.78 (quint, 2H, J=7.0 Hz), 1.65(s, 3H), 1.61 (s, 3H), 1.60 (s, 6H).

C. (E,E)-16-Bromo-2,6,10-trimethyl-2,6,10-tetradecatriene

To a solution of 2.56 g (7.79 mmol) of Part B mesylate in 15 mL of THFat room temperature was added 2.02 g (23.37 mmol) of anhydrous lithiumbromide, resulting in a mild exotherm. For this purpose, lithium bromidewas dried at 100° C. over P₂ O₅ under vacuum. The suspension was allowedto stir for 23 hours at room temperature, when it was diluted withdiethyl ether, washed with water (two portions) and brine, dried (MgSO₄)and evaporated to provide 2.29 g of a pale yellow liquid. Flashchromatography on 65 g of silica gel eluted with petroleum ether gave2.22 g (91%) of title bromide as a colorless liquid.

TLC Silica gel (hexane) R_(f) =0.35.

IR(neat) 2965, 2926, 2856, 1666, 1440, 1383, 1249, 1109 cm⁻¹.

¹ H NMR(CDCl₂, 270 MHz): δ 5.10 (br, 3H), 3.39 (t, 2H, J=6.5 Hz),1.8-2.3 (m, 12H), 1.68 (s, 3H), 1.63 (s, 3H), 1.60 (s, 6H) ppm.

Mass Spec (CI-CH₄ /N₂ O, + ions) m/e 315, 313 (M+H), 313, 311 (M+H-H₂).

D.(E,E)-[Ethoxy(5,9,13-trimethyl-4,8,12-tetradecatrienyl)phosphinyl]formicacid, ethyl ester

To a stirred suspension of 200 mg (8.22 mmol) of Mg turnings in 3 mL ofdiethyl ether under argon was added 30 μL (0.35 mmol) of1,2-dibromoethane, followed 10 minutes later by the dropwise addition of1.26 g (4.02 mmol) of Part C bromide in 5 mL of diethyl ether over 25minutes. The mixture was stirred for an hour while occasionally warmingto reflux with a warm water bath. The mixture was then sonicated for 40minutes, followed by stirring at room temperature for an additionalhour. The Grignard solution was cannulated into an addition funnel of asecond apparatus, fitted with a glass wool plug above the stopcock toremove any particulates in the solution. The Grignard solution was addeddropwise over 30 minutes to a stirred solution of 0.58 mL (4.02 mmol) ofdiethyl chlorophosphite in 5 mL of diethyl ether at 0° C. The resultingsuspension was stirred at 0° C. for 15 minutes followed by roomtemperature for one hour. The suspension was cannulated into a Schlenkfilter packed with dry Celite and filtered under a positive argonpressure. The solids were washed with 15 mL of diethyl ether and thecloudy filtrate was treated with 3.8 mL (40.0 mmol) of distilled ethylchloroformate. After stirring for 18 hours at room temperature, thesolvents were evaporated and the residue was partitioned between ethylacetate and saturated NaHCO₃. The organic layer was washed withsaturated NaHCO₃ and brine, dried (MgSO₄) and evaporated to provide 1.44g of a pale yellow oil. Flash chromatography on 175 g of silica geleluted with 25:75 ethyl acetate:petroleum ether gave 1.08 g (68%) oftitle compound as a colorless liquid.

TLC Silica gel (50:50 ethyl acetate:hexane) R_(f) =0.41.

IR(neat) 2965, 2927, 2873, 2858, 1715, 1448, 1386, 1248, 1200, 1032cm⁻¹.

¹ H NMR(CDCl₃, 270 MHz): δ 5.09 (br, 3H), 4.32 (t, 2H, J=7.5 Hz), 4.28(m, 2H), 1.8-2.2 (m, 12H), 1.68 (s, 3H), 1.60 (s, 9H), 1.36 (m, 6H) ppm.

Mass Spec (CI-CH₄ /N₂ O, + ions) m/e 398 (M+H), 325, 285, 263.

EXAMPLE 2(E,E)-[Hydroxy(5,9,13-trimethyl-4,8,12-tetradecatrienyl)phosphinyl]formicacid, disodium salt

To a stirred solution of 574.4 mg (1.44 mmol) of Example 1 compound in 5mL of dry dichoromethane at -18° C. under argon was added 0.19 mL (1.44mmol) of 2,4,6-collidine followed by 0.41 mL (2.88 mmol) ofiodotrimethylsilane. The reaction was allowed to stir for one hour at-18° C. and 1.5 hours at 0° C. An additional portion ofiodotrimethylsilane (0.10 mL, 0.70 mmol) was added and after anadditional 30 minutes at 0° C., the solvents were evaporated and theresidue was pumped under vacuum. The residue was dissolved in 7.5 mL(7.5 mmol) of 1 M NaOH and the solution was stirred for two hours atroom temperature followed by two hours at 65° C. The aqueous solutionwas lyophilized and the crude tan powder was chromatographed on a 2.5 cmdiameter×20 cm height column of CHP20P packed in water. The column waseluted with water (fractions 1-5) followed by a gradient created by theaddition of 600 mL of 70:30 acetonitrile:water to 600 mL of water,collecting approximately 10 mL fractions. Fractions 48-58 were combinedand lyophilized to provide 374.7 mg (65%) of title compound as a whitepowder.

TLC Silica gel (7:2:1 n-C₃ H₇ OH:con NH₃ :H₂ O) R_(f) =0.47.

IR(KBr) 3433 (br), 2967, 2928, 2859, 1573, 1448, 1383, 1366, 1189, 1057cm⁻¹.

¹ H NMR(D₂ O, 400 MHz): δ 5.17 (t, lH, J=7 Hz), 5.07 (m, 2H), 1.9-2.1(m, 10H), 1.60 (s, 3H), 1.57 (s, 3H), 1.53 (s, 3H), 1.52 (s, 3H), 1.47(m, 3H) ppm.

³¹ P NMR (D₂ O, 109 MHz): 6 27.95 (singlet) ppm.

Mass Spec (FAB, + ions) m/e 425 (M+2Na-H), 409 (M+Na), 387 (M+H).

Anal. Calc'd for C₁₈ H₂₉ PO₄ Na₂ (MW 386.42): C, 55.94; H, 7.58; P, 8.02

Found*: C, 56.00; H, 7.49; P, 8.27.

EXAMPLE 3(E,E)-[Hydroxy(5,9,13-trimethyl-4,8,12-tetradecatrienyl)phosphinyl]formicacid, ethyl ester, monopotassium salt

To a stirred solution of 498.5 mg (1.25 mmol) of Example 1 compound in 3mL of dry dichloromethane at 0° C. under argon was added 0.17 mL (1.29mmol) of 2,4,6-collidine followed by 0.36 mL (2.50 mmol) ofiodotrimethylsilane, dropwise over 2 minutes. The colorless solution wasallowed to stir at 0° C. for 1.7 hours, the solvent was evaporated andthe residue was pumped at high vacuum for 30 minutes. The oil wastreated with 0.26 mL (1.86 mmol) of triethylamine in 2 mL of water for10 minutes, and the mixture was partitioned between dichloromethane and1 M HCl. The aqueous layer was reextracted with dichloromethane, and thecombined organic layers were washed with brine, dried (MgSO₄) andevaporated to provide 481 mg of a colorless oil. The crude acid wasdissolved in 3 mL of methanol and treated with 1.25 mL of 1 M KOH togive a solution with a pH of 11. The methanol was evaporated and theaqueous solution was applied to a 20 cm long, 2.5 cm diameter column ofCHP20P packed in water and eluted with a gradient created by the gradualaddition of 600 mL of acetonitrile to 600 mL of water, collecting 8 mLfractions. Fractions 43-47 were combined, the acetonitrile evaporatedand the aqueous solution freeze-dried. The residue was further dried athigh vacuum to provide 274.4 mg (54%) of title compound as a dense,white lyophilate.

TLC Silica gel (7:2:1 n-C3H70H:con NH3:H20) R_(f) =0.60 or (8:1:1 CH₂Cl₂ CH₃ OH:CH₃ CO₂ H) R_(f) =0.30.

IR (KBr) 2970, 2924, 2850, 1690, 1450, 1384, 1211, 1072 cm⁻¹.

¹ H NMR (1:1 CD₃ OD:D₂ O, 400 MHz): 6 5.10 (t, 1H, J=7 Hz), 5.06 (t, 1H,J=7 Hz), 5.04 (t, 1H, J=7 Hz), 4.19 (q, 2H, J=7 Hz), 2.02, 1.94 (two m,10 H), 1.69 (m, 2H), 1.61 (s, 3H), 1.57 (s, 3H), 1.54 (s, 6H) ppm.

Mass spec (FAB,+ions) m/e 447 (M+K), 409 (M+H).

Anal. Calc'd for C₂₀ H₃₄ O₄ P₂ K: C, 58.80; H, 8.39; P, 7.58 Found*: C,59.91; H, 8.67; P, 7.64.

EXAMPLE 4(E,E)-[Hydroxy[(5,9,13-trimethyl-4,8,12-tetradecatrienyl)oxy]phosphinyl]formicacid, disodium salt

To a solution of 687 mg (3.6 mmol, 3 eq.) of (ethoxycarbonyl)phosphonicdichloride (preparation as described in Vaghefi, M. M.; McKernan, P. A.;and Robins, R. K.; J. Med. Chem., (1986), 29, 1389) in 6 mL of drytetrahydrofuran (THF) at -30° C. (CCl₄ --CO₂) under argon was addeddropwise a solution of 301 mg (1.2 mmol) of Example 1 Part Abishomofarnesol in 2 mL of dry THF over 5 minutes. After stirring for1.5 hours, the -30° bath was replaced with a 0° C. bath and the reactionwas immediately quenched with 9.6 mL (19.2 mmol, 16 eq.) of 2M NaOH. Themixture was stirred for two hours at room temperature, the THF wasevaporated, and the aqueous mixture remaining was lyophilized.Purification was carried out by chromatography on a 2.5 cm diameter×18cm height column of CHP20P packed in water. The crude compound wasloaded as a suspension in 5 mL of water. The column was eluted with 200mL of water, then 600 mL of 30:70 CH₃ CN:H₂ O, collecting 8-10 mLfractions every 1.5 minutes. Fractions 36-41 were combined, evaporated,lyophilized, and pump-dried overnight to obtain 214.1 mg (53%) of titlecompound as a white lyophilate.

TLC Silica gel (7:2:1 n-C₃ H₇ OH:NH₃ :H₂ O) R_(f) =0.47.

IR(KBr) 3500, 2964, 2925, 2890, 2856, 1581, 1447, 1373, 1227, 1091,1051, 852, 619, 586 cm⁻¹.

¹ H NMR (D₂ O, 400 MHz): δ 5.19 (t, 1H, J=6.6 Hz), 5.11 (m, 2H), 3.87(dt, 2H, J_(HH) =6.6, J_(HP) =7.0 Hz), 1.9-2.1 (m, 10H), 1.67 (m, 2H),1.64 (s, 3H), 1.62 (s, 3H), 1.57 (s, 6H) ppm.

Mass Spec (FAB,+ions) m/e 425 (M+Na⁺), 403 (M+H), 397 (M+2H-CO₂ +K⁺),381 (M+2H-Na), 359 (M+H-CO₂).

Anal. Calc'd for C₁₈ H₂₉ O₅ P.Na₂ : C, 53.73; H, 7.26; P, 7.70 Found: C,53.91; H, 6.81; P, 7.82.

The analytical sample was pig-dried at 50° C. for six hours.

EXAMPLE 5(E,E)-[Ethoxy[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]methyl]phosphinyl]formicacid, ethyl ester A.Tributyl[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]methyl]tin

A 1.15 g (10.0 mmol) of 35% KH in mineral oil was washed with three 5 mLportions of dry hexane and suspended in 15 mL of dry tetrahydrofuran(THF) under argon. A solution of 242 mg (0.95 mmol) of iodine in 10 mLof THF was added over 50 minutes and the reaction was allowed to stirfor an additional 40 minutes at room temperature to give a whitesuspension. (E,E)-Farnesol (1.5 g, 6.75 mmol) in 10 mL of THF was addeddropwise over 15 minutes, and after 75 minutes, a solution of 2.43 g(5.64 mmol) of tributyl(iodomethyl)tin was added rapidly in 6 mL of THF.The mixture was stirred for three hours and carefully quenched withmethanol. Upon dilution with diethyl ether, the organic layer was washedwith water and brine, dried (MgSO₄) and evaporated to give 3.21 g of apale yellow liquid. The crude material was chromatographed on 720 g ofsilica gel eluted with 0.5:99.5 diethyl ether: petroleum ether, and theimpure fractions were further purified on 150 g of silica gel elutedwith 0.33:99.67 diethyl ether:petroleum ether to provide a total of 2.09g (71%) of pure title stannane as a colorless liquid.

TLC Silica gel (2:98 diethyl ether:hexane) R_(f) =0.4.

IR(CCl₄) 2958, 2926, 2872, 2854, 1464, 1456, 1377, 1053 cm⁻¹.

¹ H NMR(CDCl₃, 270 MHz): δ 5.32 (t, 1H, J=6.5 Hz), 5.12 (t, 1H, J=7.0Hz), 5.09 (t, 1H, J=7.0 Hz), 3.87 (d, 2H, J=6.5 Hz), 3.72 (s, 2H), 2.05(m, 8H), 1.68 (s, 3H), 1.66 (s, 3H), 1.60 (s, 6H), 1.52 (quint, 6H,J=8.0 Hz), 1.30 (sextet, 6H, J=8.0 Hz), 0.90 (m, 15H) ppm.

B.(E,E)-[Ethoxy[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]methyl]phosphinyl]formicacid, ethyl ester

To a solution of 855.8 mg (1.63 mmol) of Part C stannane in 11 mL of drydiethyl ether at -78° C. under argon was added in 1.12 mL (1.80 mmol) ofn-butyllithium (1.6M in hexane) over 5 minutes. After 30 minutes at -78°C., 0.80 mL (5.53 mmol) of diethyl chlorophosphite was added as rapidlyas possible. The cold bath was allowed to warm to room temperature verygradually over 3.5 hours, and the white suspension was allowed to stirfor an additional 30 minutes at room temperature. Freshly distilledethyl chloroformate (2.4 mL, 25.1 mmol) was added and the mixture wasallowed to stir at room temperature for 160 minutes. The volatiles wereremoved in vacuo and the residue was diluted with ethyl acetate, washedwith water (three portions), saturated NaHCO₃ and brine, dried (MgSO₄)and evaporated to provide 1.28 g of a colorless liquid. The crudeproduct was flash chromatographed on 150 g of silica gel packed in 8:92acetone:hexane and eluted with 12:88 acetone: hexane to provide 418.2 mg(64%) of pure title compound as a colorless oil.

TLC Silica gel (20:80 acetone:hexane) R_(f) =0.24.

IR(CCl₄) 2965, 2930, 2865, 1719, 1445, 1383, 1264, 1239, 1199, 1095,1032, 965 cm⁻¹.

¹ H NMR(CDCl₃, 270 MHz) δ 5.30 (t, 1H, J=7.0 Hz), 5.08 (m, 2H), 4.34 (qon m, 4H, Jq=6.0 Hz), 4.14 (d, 2H, J=7.0 Hz), 3.95 (ABX, 2H, J_(AB) =14Hz, J_(AX) =7.0 Hz, J_(BX) =5.8 Hz), 2.08 (s, 8H), 1.68 (s, 6H), 1.60(s, 6H), 1.40 (t, 3H, J=7.0 Hz), 1.36 (t, 3H, J=7.0 Hz) ppm.

Mass Spec (CI-CH₄ /N₂ O, +ions) m/e 401 (M+H), 429 (M+C₂ H₅), 441 (M+C₃H₅).

EXAMPLE 6(E,E)-[Hydroxy[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]methyl]phosphinyl]formicacid, disodium salt

A 490.4 mg (1.22 mmol) sample of Example 5 diester was stirred with 12mL of 1M NaOH and 8 mL of distilled water for 1.5 hours at roomtemperature followed by 2.5 hours at 70° C. to give a homogeneoussolution. Lyophilization gave a dense residue which was difficult toredissolve in water. The residue was heated to 90° C. in 10 mL ofdistilled water for 1 hour resulting in a homogeneous solution, whichupon cooling to room temperature deposited a small amount of insolublematerial. This solution was loaded onto a 2.5 cm diameter, 20 cm heightcolumn of CHP20P packed in water. The column was eluted with water(fractions 1-13), followed by a gradient created by the addition of80:20 acetonitrile:water to a reservoir of 500 mL of water, such as tomaintain the reservoir level at 500 mL (fractions 14-42). The additionof the acetonitrile mixture was discontinued after fraction 42 and theelution was continued isocratically. Approximately 10 mL fractions werecollected. Fractions 38-50 were combined, the acetonitrile wasevaporated and the aqueous remainder was freeze-dried to provide 337.3mg (71%) of pure title compound as a fluffy-white lyophilate. Thelyophilate was dried further at room temperature under high vacuumovernight. (Fractions 51-58 were similarly processed to provide 122 mg(25%) of slightly impure title compound.

TLC Silica gel (7:2:1 n-C₃ H₇ OH:con NH_(3:) H₂ O) R_(f) =0.48.

IR(KBr) 3430, 2967, 2924, 2857, 1597, 1449, 1385, 1362, 1189, 1042, 764cm⁻¹.

¹ H NMR(D 0, 400 MHz) δ 5.37 (t, 1H, J=7.0 Hz), 5.15 (t, 1H, J=6.5 Hz),5.12 (t, 1H, J=7.0 Hz), 4.11 (d, 2H, J=7.0 Hz), 3.70 (d, 2H, J=6.5 Hz),2.10 (m, 6H), 1.98 (t, 2H, J=7.0 Hz), 1.69 (s, 3H), 1.65 (s, 3H), 1.59(s, 3H), 1.58 (s, 3H) ppm.

Mass Spec (FAB, +ions) m/e 411 (M+Na), 367 (M+Na-CO₂), 345 (M+H-CO₂).

Anal. Calc'd for C₁₇ H₂₇ PNa₂ O₅ : C, 52.58; H, 7.01; P, 7.98 Found: C,52.51; H, 7.27; P, 8.28.

EXAMPLE 7(E,E)-[Ethoxy(6,10,14-trimethyl-5,9,13-pentadecatrienyl)phosphinyl]formicacid, ethyl ester A. 6,10,14-Trimethyl-5,9,13-pentadecatrien-1-ol (1)1-Chloro-3,7,11-trimethyl-2,6,10-dodecatriene

(Note all temperatures indicated are for the contents of the reactionflask). To a stirred solution of 299 mg (2.24 mmol) ofN-chlorosuccinimide in 15 mL of dichloromethane at -30° C. under argonwas added 0.18 mL (2.45 mmol) of distilled dimethyl sulfide over 5minutes. After 10 minutes at -30° C., the reaction was allowed to warmto 0° C. for 10 minutes, followed by cooling to -40° C. A solution of441.4 mg (1.99 mmol) of 3,7,11-trimethyl-2,6,10-tridecatrien-1-ol in 5mL of dichloromethane was added dropwise over 10 minutes. The reactionwas allowed to warm gradually to 0° C. over 1 hour, and then maintainedat 0° C. for 1 hour. After quenching with cold water, the mixture wasextracted with hexane and the hexane extract was washed with cold waterand cold brine, dried (MgSO₄) and evaporated to afford 483 mg of a crudeproduct. Rapid flash chromatography on 20 g of Merck 9385 silica geleluted with 3:97 ethyl acetate:petroleum ether provided 406.5 mg (85%)of a colorless liquid. ¹³ C NMR indicated that this material contained atrace (3%) impurity.

TLC:Silica gel (2:98 ethyl acetate:hexane) R_(f) =0.56.

¹ H NMR(CDCl₃, 270 MHz): δ 5.44 (t, 1, J=7.9 Hz), 5.09 (t, 2, J=5.8 Hz),4.07 (d, 2, J=7.9 Hz), 1.9-2.2 (m, 9), 1.72 (s, 3), 1.68 (s, 3), 1.60(s, 6) ppm.

(2) Dichloro[mu-[1-propanolato(2-)-C³ :O¹ ]]dimagnesium

A modification of the procedure of G. Cahiez et al was employed(Tetrahedron Letters, 1978, 3013-4): To a stirred solution of 1.89 g (20mmol) of 3-chloropropanol in 20 mL of THF under argon at -20° C. wasadded 10 mL (20 mmol) of 2M phenylmagnesium chloride in THF over 15minutes. After 10 minutes at -20° C., the reaction was allowed to warmto room temperature, 730 mg (30 mmol) of magnesium turnings were addedand the reaction was heated to reflux. Two 40 μL portions of1,2-dibromoethane were added, the first portion injected at the start ofreflux, and the second after 1 hour. After refluxing for a total of 2hours, the reaction was allowed to cool to room temperature and wasdiluted with 37 mL of THF for a theoretical concentration of 0.3M.

(3) 6,10,14-Trimethyl-5,9,13-pentadecatrien-1-ol

A solution of 37.5 mL (20.3 mmol, 5.1 eq.) of a 0.54M solution ofGrignard reagent (Part (2)) in tetrahydrofuran and 9 mL ofhexamethylphosphoramide at room temperature under argon was treated over10 minutes with a solution of 955.5 mg (3.97 mmol) of farnesyl chloride(Part (1)) in 5 mL of tetrahydrofuran. After one hour, the reactionmixture was diluted with a mixture of 1:1 diethyl ether:hexane andquenched with 1M HCl. The organic phase was washed with three 25 mLportions of saturated NaHCO₃, three 25 mL portions of H₂ O, and 25 mL ofbrine, dried over MgSO₄ and evaporated to obtain 995.0 mg of crudeproduct. Purification required two chromatographies. The first was runon 70 g of silica gel, eluting with 1:99 ethyl acetate:CH₂ Cl₂ toprovide 484.3 mg of impure material and 307.7 mg of pure title compound.The second chromatography, of the impure fractions, on 50 g of silicagel eluted with 0.75:99.25 ethyl acetate:CH₂ Cl₂ gave 117.2 mg ofslightly impure material and 302.8 mg of pure title compound.Combination of pure material from both columns gave a yield of 610.5 mg(58%) of pure desired title isomer.

TLC:Silica gel (10:90 diethyl ether:CH₂) R_(f) =0.38.

IR (CCL₄) 3639, 3450, 2964, 2930, 2858, 1449, 1382, 1058, 1028, 776, 750cm⁻¹.

¹ H NMR(CDCl₃, 270 MHz): δ 5.10 (m, 3H), 3.62 (t, 2H, J=6.5 Hz), 2.00(m, 10H), 1.69 (s, 3H), 1.61 (s, 9H), 1.2-1.7 (m, 5H, OH) ppm.

Mass Spec (CI-CH₄ /N₂ O, +ions) m/e 282 (M+NH₄), 265 (M+H), 263(M+H-H₂).

B. (E,E)-15-Iodo-2,6,10-trimethyl-2,6,10-pentadecatriene

To a stirred solution of 363.8 mg (1.38 mmol) of Part A alcohol in 6 mLof dichloromethane at 0° C. was added 0.39 mL (2.76 mmol) oftriethylamine followed by the dropwise addition of 0.14 mL (2.76 mmol)of methanesulfonyl chloride over 5 minutes. After stirring for 1 hour at0° C., the mixture was diluted with diethyl ether and the organic phasewas washed with 10% HCl, water, saturated NaHCO₃ and brine, dried(MgSO₄) and evaporated to give 458.8 mg of the mesylate as a colorlessoil.

The crude mesylate was dissolved in 10 mL of acetone, treated with 414mg (2.76 mmol) of sodium iodide and heated to 40° C. under argon for 17hours. The mixture was diluted with hexane, washed with water, 4% sodiumthiosulfate, water and brine, dried (MgSO₄), and evaporated to provide acolorless oil. Flash chromatography on 30 g of silica gel eluted withhexane gave 466.6 mg (90%) of the pure title iodide as a colorless oil.

TLC Silica gel (hexane) R_(f) =0.32.

IR (CCl₄) 2965, 2927, 2854, 1449, 1381, 1222, 809 cm⁻¹.

¹ H NMR(CDCl₃, 270 MHz): δ 5.10 (m, 3H), 3.18 (t, 2H, J=7 Hz), 2.00 (m,10H), 1.82 (quint, 2H, J=7 Hz), 1.68 (s, 3H), 1.60 (s, 9H), 1.44 (m, 2H)ppm.

Mass Spec (CI-CH_(4/) N₂ O, +ions) m/e 392 (M+NH₄), 375 (M+H).

C.(E,E)-[Ethoxy(6,10,14-trimethyl-5,9,13-pentadecatrienyl)phosphinyl]formicacid,ethyl ester

A solution of 266.4 mg (0.712 mmol) of the Part B iodide in 4 mL ofdiethyl ether was added dropwise over 20 minutes to a solution of 1 mL(1.7 mmol) of 1.7M t-butyllithium in pentane in 3 mL of diethyl ether at-78° C. under argon. After 45 minutes at -78° C., the solution wasallowed to warm gradually to room temperature and stir for 1 hour. Theanion was transferred via cannula with the aid of 2 mL of diethyl etherto the addition funnel of a second apparatus. This solution was addeddropwise over 50 minutes to a solution of 0.23 mL (1.58 mmol) of diethylchlorophosphite in 4 mL of diethyl ether at -78° C. After 50 minutes at-78° C., the reaction was allowed to warm gradually to room temperatureover 2 hours and then stir at room temperature for 1 hour. Excessdistilled ethyl chloroformate (0.7 mL, 7.32 mmol) was added, and thereaction was allowed to stir at room temperature for 16 hours. The whitesuspension was evaporated and the residue was dissolved in ethylacetate, washed with saturated NaHCO₃, water and brine, dried (MgSO₄)and evaporated to provide 351 mg of a crude oil. The crude product wasflash chromatographed on 40 g of silica gel eluted with 20:80 ethylacetate:hexane to provide 169 mg (58%) of pure title compound as acolorless oil.

TLC Silica gel (50:50 ethyl acetate:hexane) R_(f) =0.33.

IR(CCl₄) 2979, 2928, 2856, 1712, 1445, 1251, 1196, 1032, 960 cm⁻¹.

¹ H NMR(CDCl₃, 270 MHz): δ 5.03 (m, 3H), 4.25 (q, 2H, J=7.5 Hz), 4.20(m, 2H), 1.93 (m, 12H), 1.62 (s, 3H), 1.52 (s, 9H), 1.49-1.61 (m, 2H),1.38 (quint, 2H, J=7.5 Hz), 1.43, 1.44 (two t, 6H, J=7.5 Hz) ppm.

Mass Spec (CI-CH₄ /N₂ O, +ions) m/e 453 (M+C₃ H₅), 441 (M+CH₂ H₅), 413(M+H).

EXAMPLE 8(E,E)-[Hydroxy(6,10,14-trimethyl-5,9,13-pentadecatrienyl)phosphinyl]formicacid, disodium salt

To a stirred solution of 163.8 mg (0.397 mmol) of Example 7 ester in 2mL of dry dichloromethane at 0° C. under argon was added 60 μL (0.45mmol) of 2,4,6-collidine followed by 120 μL (0.843 mmol) ofiodotrimethylsilane. After 1.5 hours at 0° C., the solution wasevaporated, the residue was evaporated with benzene and pumped at highvacuum. This material was dissolved in 2.5 mL of 1M NaOH and stirred for3 hours at room temperature followed by 2 hours at 65° C. The solutionwas lyophilized and the crude product was purified by MPLC on a 19 cmheight, 2.5 cm diameter column of CHP20P. The column was packed andeluted with water (fractions 1-6), followed by a gradient created by thegradual addition of 75:25 acetonitrile:water to a reservoir of water,maintaining the reservoir volume at 500 mL. Fractions 31-38 werecombined, the acetonitrile was evaporated and the residue wasfreeze-dried to give 84.4 mg (53%) of pure title product as a whitelyophilate, which was dried further at high vacuum overnight.

TLC Silica gel (7:2:1 n-C₃ H₇ OH:con NH_(3:) H₂ O) R_(f) =0.41

IR(KBr) 3450, 2966, 2927, 2857, 1574, 1448, 1382, 1365, 1184, 1112, 1054cm⁻¹.

¹ H MR(D₂ O, 400 MHz): δ 5.18 (t, 1H, J=7 Hz), 5.12 (t, 1H, J=7 Hz),5.10 (t, 1H, J=8 Hz), 2.04 (m, 10H), 1.64 (s, 3H), 1.60 (s, 3H), 1.56(s, 6H), 1.5-1.7 (m, 2H), 1.49 (m, 2H), 1.37 (quint, 2H, J=7 Hz) ppm.

Mass Spec (FAB+ions) m/e 423 (M+Na), 401 (M+H), 379 (M+2H-Na), 357(M+H-CO₂), 335 (M+2H-Na-CO₂).

Anal. Calc'd for C₁₉ H₃₁ PNa₂ O₄.0.38 eq H₂ O: C, 56.04; H, 7.86; P,7.61 Found: C, 56.04; H, 7.87; P, 7.67.

EXAMPLE 9(E,E)-[Hydroxy[(4,8,12-trimethyl-3,7,11-tridecatrienyl)oxy]phosphinyl]formicacid, disodium salt A. (E,E)-4,8,12-Trimethyl-3,7,11-tridecatrien-1-ol(1) (E,E)-3,7,11-Trimethyl-2,6,10-dodecatrienaldehyde [(E,E)-Farnesal]

A solution of oxalyl chloride (4.68 g, 0.037 mol) in dry CH₂ Cl₂ underargon atmosphere was cooled to -65° C. A solution of dimethyl sulfoxide(DMSO) (5.33 ml) in CH₂ Cl₂ (17 ml) was added rapidly, dropwise, to thecooled oxalyl chloride solution. After stirring for 7 minutes at -65°C., a 10 ml CH₂ Cl₂ solution of (E,E)-farnesol (7.0 g, 0.032 mol) wasadded over 10 minutes to the reaction solution at -65° C.: a precipitateformed upon the addition of approximately half of the farnesol solution.After the addition of the farnesol solution was completed, the reactionwas stirred at -65° C. for 25 minutes, and then 22.4 ml (2 mol) oftriethylamine was added over 10 minutes. After stirring for anadditional 15 minutes at -65° C., the reaction was warmed to roomtemperature, and then diluted with water (˜200 ml). The resultingaqueous layer was extracted several times with CH₂ Cl₂. The combinedorganic layers were washed once with saturated aqueous NaCl solution,once with 1% HCl, once with 5% Na₂ CO₃ solution and once with saturatedaqueous NaCl solution. The resulting organic layer was dried over MgSO₄to give 7.05 g (100%) of a clear oil after filtration and solventremoval.

TLC Silica gel (20% ethyl acetate/hexane) R_(f) =0.34.

¹ H NMR (CDCl₃, 270 MHz) 69.98 (d, 1H, J=7 Hz), 5.88 (broad d, 1H, J=7Hz), 5.08 (m, 2H), 2.22 (m, 4H), 2.17 (s, 3H), 2.02 (m, 4H), 1.66 (s,3H), 1.60 (s, 6H) ppm.

¹³ C-NMR (CDCl₃, 67.8 MHz) 6 191.0, 163.6, 136.5, 131.3, 127.4, 124.0,122.4, 40.5, 39.6, 26.6, 25.6, 17.6, 17.5, 15.9 ppm.

(2) 4,8,12-Trimethyl-1,3,7,11-tridecatetraene

A suspension of methyltriphenylphosphonium iodide (8.07 g, 0.02 mole) in61 ml of dry tetrahydrofuran (THF), under argon atmosphere was cooled to0° C. To this suspension at 0° C. was added 9 mL (18 mmol) ofphenyllithium (2.0M in diethyl ether/hexane 30:70) over 10 minutes.After the addition was complete, the reaction mixture containing excessphosphonium salt was warmed to room temperature and stirred for 40minutes. The reaction mixture was then recooled to 0° C., and a 10 mlTHF solution of the Part (1) aldehyde (4.0 g, 0.018 mol) was added over12 minutes. After stirring for 10 minutes at 0° C., the reaction waswarmed to room temperature. The reaction was quenched with CH₃ OH after2 hours at room temperature. The THF was removed from the reactionmixture to give a slurry which was triturated with petroleum ether, andsubsequently, filtered through a Celite pad in a sintered glass funnel.The solids were then boiled in petroleum ether and refiltered as above.The resulting yellow oil was passed through 50 g of Florisil (100-200mesh) eluted with ˜400 ml of petroleum ether providing the titletetraene (3.36 g, 86%) as a clear oil after solvent removal.

TLC Silica gel (20% ethyl acetate/hexane) R_(f) =0.68.

¹ H NMR (CDCl₃, 270 MHz) δ 6.56 (ddd, 1H, J=17, 12, 6 Hz), 5.85 (d, 1H,J=12 Hz), 5.10 (m, 2H), 5.02 (m, 2H), 2.05 (m, 8H), 1.75 (s, 3H), 1.67(s, 3H), 1.60 (s, 6H) ppm.

¹³ C-NMR (CDCl₃, 67.8 MHz) δ 139.3, 135.3, 133.4, 131.2, 125.5, 124.3,123.9, 114.5, 39.9, 39.7, 26.8, 26.4, 25.6, 17.7, 16.6, 15.9 ppm.

(3) (E,E)-4,8,12-Trimethyl-3,7,11-tridecatrien-1-ol

Neat 2-methyl-2-butene (2.25 g, 0.032 mol) was added to a 1.0M BH₃ -THFsolution (16.9 ml) at -50° C. and under argon. After the addition wascomplete, the reaction was stirred for 2 hours at 0° C. The resultingdisiamylborane solution was transferred via cannula over 1 hour to aflask containing a 17 ml THF solution of Part A(2) tetraene (3.36 g,0.015 mol) under argon atmosphere and cooled to 0° C. After the transferwas complete, the reaction was allowed to gradually warm to roomtemperature, and then it was stirred overnight at room temperature. Thereaction mixture was cooled to 0° C., and 5.1 ml of 3N NaOH was addedrapidly. After stirring for 10 minutes, the reaction mixture was cooledin an ice-salt bath and 5.1 ml of 30% H₂ O₂ was added dropwise.Subsequently, the reaction was warmed to room temperature and stirredfor 4 hours after which it was diluted with H₂ O, and the resultingaqueous layer was extracted several times with diethyl ether. Thecombined organic layers were dried over MgSO₄. Purification by flashchromatography eluting with 20% ethyl acetate/hexane provided the titlealcohol (2.62 g, 74%) as a clear oil.

TLC Silica gel (20% ethyl acetate/hexane) R_(f) =0.23.

IR (Film) 3340 (br), 2965, 2920, 1665, 1440, 1380, 1100, 1050 cm⁻¹.

¹ H NMR (CDCl₃, 270 MHz) δ 5.10 (m, 3H), 3.61 (t, 2H, J=6 Hz), 2.29 (q,2H, J=6 Hz), 2.03 (m, 8H), 1.67 (s, 3H), 1.65 (s, 3H), 1.60 (s, 6H) ppm.

¹³ C NMR (CDCl₃, 67.8 MHz) δ 138.8, 135.2, 131.2, 124.3, 123.9, 119.9,62.4, 39.8, 39.7, 31.5, 26.7, 26.5, 25.6, 17.6, 16.1, 15.9 ppm.

B.(E,E)-[Hydroxy[(4,8,12-trimethyl-3,7,11-tridecatrienyl)oxy]phosphinyl]formicacid, disodium salt

To a solution of 967.5 mg (5.08 mmol, 3 eq.) of(ethoxycarbonyl)phosphonic dichloride (preparation as described inVaghefi, M. M.; McKernan, P. A.; and Robins, R. K.: J. Med. Chem.,(1986), 29, 1389) in 10 mL of dry THF at -30° C. (CCl₄ -CO₂) under argonwas added dropwise a solution of 400.2 mg (1.7 mmol) of Part Ahomofarnesol in 4 mL of dry THF over 8 minutes. After stirring for twohours, the -30° C. bath was replaced with a 0° C. bath and the reactionwas immediately quenched with 13.6 mL (27.2 mmol, 16 eq.) of 2 N NaOH.The mixture was stirred for three hours at room temperature, the THF wasevaporated, and the aqueous mixture remaining was lyophilized.Purification was carried out by chromatography on a 2.5 cm diameter×18cm height column of CHP20P packed in water. The crude compound wasloaded as a suspension in 6 mL of water. The column was eluted with 250mL of water, then 600 mL of 30:70 CH₃ CN:H₂ O, collecting 8-10 mLfractions every 1.5 minutes. Fractions 40-44 were combined, theacetonitrile was evaporated, and the aqueous solution was lyophilized.The residue was pump-dried overnight to obtain 341.5 mg (52%) of afluffy, white lyophilate.

TLC Silica gel (7:2:1 n-C₃ H₇ OH:NH₃ :H₂ O) R_(f) =0.38.

IR(KBr) 3440, 2967, 2925, 2856, 1581, 1447, 1424, 1372, 1227, 1093,1073, 1034, 847, 798, 774 cm⁻¹.

¹ H NMR(D₂ O, 400 MHz) δ 5.18 (t, 1H, J=6.6 Hz), 5.12 (m, 2H), 3.82 (q,2H, J_(HH) =7.3 Hz, J_(HP) =7.3 Hz), 3.37 (dt, 2H, J=6.6, 7.3 Hz),1.9-2.1 (m, 8H), 1.65 (s, 6H), 1.58 (s, 3H), 1.57 (s, 3H) ppm.

Mass Spec (FAB,+ions) m/e 411 (M+Na), 389 (M+H), 383 (M+2H+K-2Na), 367(M+2H-Na), 345 (M+H-CO₂), 281.

Anal. Calc'd for C₁₇ H₂₇ O₅ P.Na₂ : C, 52.58; H, 7.01; P, 7.98 Found: C,52.76, H, 6.92; P, 7.77.

The analytical sample was dried at 50° C. for six hours.

EXAMPLE 10(E,E)-[Hydroxy[(4,8,12-trimethyl-3,7,11-tridecatrienyl)oxy]phosphinyl]formicacid, ethyl ester, monosodium salt

To a solution of 965.3 mg (5.08 mmol, 3 eq.) of(ethoxycarbonyl)phosphonic dichloride in 10 mL of dry THF at -30° C.(CCl₄ -CO₂) under argon was added dropwise over seven minutes a solutionof 397.1 mg (1.7 mmol) of Example 9, Part A homofarnesol in 4 mL of THF.After stirring for 2.5 hours at -30° C. the cold batch was removed, thereaction was immediately quenched with 2 mL of water and stirringcontinued for an additional one-half hour. The mixture was diluted with40 mL of diethyl ether and washed with four 10 mL portions of water, and10 mL of brine, dried over MgSO₄ and evaporated to obtain 640.9 mg ofcrude phosphonic acid. The crude acid was dissolved in 2 mL of 1:1ethanol:H₂ O and the solution was treated with 1.7 mL (1.7 mmol, leq.)of 1M NaOH to attain pH 6.15. The ethanol was evaporated and theremaining aqueous solution was lyophilized to obtain 571.0 mg of crudetitle compound. Purification was by chromatography on a 2.5 cmdiameter×18 cm height column of CHP20P packed in water and eluted with agradient created by the gradual addition of 300 mL of CH₃ CN into 300 mLof 5:95 CH₃ CN:H₂₀. Approximately 10 mL fractions were collected every1.5 minutes. Fractions 34-39 were combined, evaporated, lyophilized andpump-dried overnight to obtain 315.1 mg (47%) of title compound as agummy white lyophilate.

TLC Silica gel (7:2:1 n-C₃ H₇ OH:con NH₃ :H₂ O) R_(f) =0.68.

IR(KBr) 2967, 2928, 2917, 2856, 1690, 1472, 1447, 1383, 1261, 1095,1064, 1021, 940, 858, 792, 785, 768, 759 cm⁻¹.

¹ H NMR(1:1 CD₃ OD:D₂ O, 400 MHz) δ 5.28 (t, 1H, J=7.0 Hz), 5.21 (m,2H), 4.36 (q, 2H, J=7.0 Hz), 4.04 (q, 2H, J=7.0 Hz), 2.49 (q, 2H, J=7.0Hz), 2.0-2.2 (m, 8H), 1.78 (s, 3H), 1.76 (s, 3H), 1.70 (s, 6H), 1.44(dt, 3H, J=1.1, 7.0 Hz) ppm.

Mass Spec (FAB,+ions) m/e 433 (M+K), 417 (M+Na), 395 (M+H), 347.

Anal. Calc'd for C₁₉ H₃₂ O₅ P.Na: C, 57.86; H, 8.18; P, 7.85 Found: C,58.37; H, 8.16; P, 7.65.

The analytical sample was dried at 50° C. for six hours.

EXAMPLE 11A(E,E)-[Ethoxy(4,8,12-trimethyl-3,7,11-tridecatrienyl)phosphinyl]formicacid, ethyl ester A. (E,E)-3,7,11-Trimethyl-2,6,10-dodecatrienyl bromide

A solution of 1.00 g (4.5 mmol) of (E,E)-farnesol in 10 mL of distilleddiethyl ether at 0° C. under argon in the dark was treated dropwise witha solution of 195 μL (2.05 mmol, 0.45 eq.) of PBr3 in 2 mL of diethylether. The resultant mixture ws stirred at 0° C. for one hour, thenquenched with water and separated. The organic phase was washed with 5mL of H₂ O, 5 mL of saturated NaHCO₃, and 5 mL of brine, dried over Na₂SO₄ and evaporated to give 1.26 (98%) of crude bromide as a clear oil.

TLC Silica (2:8 ethyl acetate:hexane) R_(f) =0.69 (decomposes).

¹ H NMR (CDCl₃) δ 5.52 (t, 1H, J=8.5 Hz), 5.08 (m, 2H), 4.01 (d, 2H),1.9-2.2 (m, 8H), 1.73 (s, 3H), 1.68 (s, 3H), 1.60 (s, 6H) ppm.

B.(E,E)-4,5-Dihydro-2-[(3,7,11-trimethyl-4,8,12-tridecatrienyl)thio]thiazole

To a stirred solution of 3.57 g (26.8 mmol) of 1-methylthio(thiazoline)in 50 mL of THF at -78° C. under argon was added 16 mL (25.6 mmol) of1.6M n-butyllithium in hexane over 15 minutes to give a yellow solution.After stirring for one hour, 6.33 g (22.4 mmol) of Part A bromide in 10mL of THF was added over 15 minutes. The reaction was allowed to stirfor one hour at -78° C., warm gradually to 0° C. for one hour, stir at0° C. for one hour followed by quenching with saturated NH₄ Cl. Themixture was extracted with diethyl ether, washed with water and brine,dried (MgSO₄) and evaporated to provide 9 g of a crude yellow liquid.This material was flash chromatographed on 500 g of silica gel, packedin 1.5:98.5 THF/petroleum ether and eluted with 3:97 THF/petroleum etherto afford 6.49 g (85%) of pure title compound as a colorless liquid.

TLC Silica gel (10:90 ethyl acetate/hexanes) R_(f) =0.29.

IR (CCl₄) 2965, 2923, 2852, 1570, 1446, 1382, 1304, 1280, 1108, 1097,1071, 1018, 996, 920 cm⁻¹.

D.(E,E)-[Ethoxy(4,8,12-trimethyl-3,7,11-tridecatrienyl)phosphinyl]formicacid, ethyl ester

To a solution of 1.7 mL (2.74 mmol) of 1.6M t-butyllithium in pentane in3 mL of diethyl ether at -78° C. under argon was added 482.4 mg (1.39mmol) of the Part C iodide in 4 mL of diethyl ether over 10 minutes.After 45 minutes at -78° C., the pale yellow suspension was allowed towarm to 0° C. for one hour, and then transferred to an addition funnelof a second apparatus via cannula under argon. The lithium reagentsolution was added dropwise over 45 minutes to 0.20 mL (1.39 mmol) ofdiethyl chlorophosphite in 4 mL of diethyl ether at 0° C. After 20minutes at 0° C. and 1.5 hours at room temperature, the white suspensionwas cannulated into a Schlenck filter, and filtered through dry Celite.The solids were washed with 15 mL of diethyl ether, and the filtrate wastreated with 1.3 mL (13.6 mmol) of distilled ethyl chloroformate. Themixture was allowed to stir for 21 hours at room temperature, thesolvents were evaporated and the residue was partitioned between ethylacetate and saturated NaHCO₃. The organic layer was washed withsaturated NaHCO₃ and brine, dried (MgSO₄), and evaporated to provide490.6 mg of a pale yellow oil. Flash chromatography on 45 g of silicagel packed in 10% THF/hexane and eluted with 20-30% THF/hexane gave192.5 mg (36%) of title ester as a colorless oil.

TLC Silica gel (30:70 THF/hexane) R_(f) =0.30.

¹ H NMR (CDCl₃) δ 5.10 (m, 3H), 4.20 (t, 2H, J=7.9 Hz), 3.36 (t, 2H,J=7.9 Hz), 3.09 (t, 2H, J=7.4 Hz), 2.39 (q, 2H, J=7.4Hz), 2.00 (m, 8H),1.68 (s, 3H), 1.62 (s, 3H), 1.60 (s, 6H) ppm.

C. (E,E)-13-Iodo-2,6,10-trimethyl-2,6,10-tridecatriene

2.98 g (8.83 mmol) of Part B compound in 6 mL of dimethylformamide (DMF)in a pressure bottle was treated with 42 mg of calcium carbonate and twodrops of mercury, and then flushed with argon. Iodomethane (12 mL, 193mmol) was added, the bottle sealed and the contents heated at 60°-70° C.for 22 hours. The mixture was diluted with an equal volume of petroleumether and the salts were removed by filtration. The volatiles wereremoved by short-path distillation at 60° C. at atmospheric pressurefollowed by aspirator pressure. The yellow residue was dissolved in 100mL of hexane and washed with 2% Na₂ S₂ O₃ (2×25 mL), water (2×25 mL),and brine, dried (MgSO₄) and evaporated to provide 2.95 g of a crudeyellow liquid. The crude material was flash chromatographed on 225 g ofsilica gel eluted with petroleum ether to provide 2.506 g (82%) of titleiodide as a colorless oil.

TLC Silica gel (hexane) R_(f) =0.33.

¹ H NMR (CDCl₃) δ 5.11 (m, 3H), 3.10 (t, 2H, J=7 Hz), 2.58 (q, 2H, J=7Hz), 2.00 (m, 8H), 1.68 (s, 3H), 1.60 (s, 9H) ppm.

IR (CCl₄) 2981, 2927, 2915, 1714, 1445, 1252, 1195, 1164, 1034, 961,859, 801 cm⁻¹.

¹ H NMR (CDCl₃) δ 5.10 (m, 3H), 4.30 (m, 4H), 2.34 (m, 2H), 2.00 (m,10H), 1.68 (s, 3H), 1.63 (s, 3H), 1.60 (s, 6H), 1.36 (m, 6H) ppm.

Mass Spec (CI-CH₄ /N₂ O,+ions) m/e 413 (M+C₂ H₅), 385 (M+H).

EXAMPLE 11B(E,E)-[Hydroxy(4,8,12-trimethyl-3,7,11-tridecatrienyl)phosphinyl]formicacid, disodium salt

To a stirred solution of 185.4 mg (0.482 mmol) of Example 11A ester in 3mL of dry CH₂ Cl₂ at 0° C. under argon was added 70 μL (0.53 mmol) of2,4,6-collidine followed by 0.15 mL (1.05 mmol) of iodotrimethylsilane.The reaction was allowed to stir for 2.5 hours at 0° C. and then thevolatiles were evaporated and the residue was pumped at high vacuum. Tothe remainder was added 2.5 mL of 1M NaOH, and the solution was stirredat room temperature for 1.5 hours followed by 80° C. for 2 hours. Afterfreeze drying, the tan crude product was purified by MPLC on CHP20P gel(2.5 cm diameter, 15 cm height), eluted with a gradient created by thegradual addition of methanol to a reservoir of 10:90 water: methanol.The appropriate fractions were combined, the methanol evaporated, andthe aqueous remainder was freeze-dried to provide 110 mg (61%) of puretitle salt as a white lyophilate.

TLC Silica gel (7:2:1 n-propanol:con NH₃ :water) R_(f) =0.49.

IR (KBr) 2967, 2924, 2856, 1574, 1447, 1182, 1053 cm⁻¹.

¹ H NMR (D₂ O, 400 MHz) δ 5.25 (t, 1H, J=7 Hz), 5.17 (m, 2H), 2.17 (m,2H), 2.01 and 2.10 (two m, 4H each), 1.67 (s, 3H), 1.63 (s, 3H), 1.60(s, 6H), 1.60-1.75 (m, 2H) ppm.

¹³ C NMR (D 0, 67.8 MHz) δ 180.9 (d, J=162.8 Hz), 136.5, 136.4, 133.0,124.8, (d, J=9 Hz), 124.5, 39.1, 39.0, 29.38 (d, J=87 Hz), 26.0, 25.1,20.7, 17.2, 15.5, 15.4 ppm.

Anal. Calc'd for C₁₇ H₂₇ O₄ PNa₂ : C, 54.83; H, 7.31; P, 8.32 Found: C,55.01; H, 7.29; P, 8.48.

The analytical sample was dried to constant weight at 50° C.

EXAMPLE 12A (E,E)-(5,9,13-Trimethyl-4,8,12-tetradecatrienyl)phosphinicacid, ethyl ester A.(E,E)-14-Iodo-2,6,10-trimethyl-2,6,10-tetradecatriene

The crude Example 1, Part B mesylate prepared from 441.1 mg (1.76 mmol)of the corresponding alcohol according to the procedure of Example 1,Part B, was dissolved in 5 mL of acetone and treated with 530 mg (3.52mmol) of sodium iodide. The reaction was allowed to stir for 16 hours atroom temperature followed by 5 hours at reflux. The suspension wasdiluted with hexane and stirred with dilute aqueous sodium bisulfite todischarge the yellow color. The organic layer was washed with water andbrine, dried (MgSO₄), and evaporated to provide 577 mg of crude product.Flash chromatography on 35 g of silica gel eluted with hexane gave 550.9mg (87%) of title iodide as a colorless liquid.

TLC Silica gel (hexane) R_(f) =0.31.

¹ H NMR (CDCl₃, 270 MHz) δ 5.09 (m, 3H), 3.16 (t, 2H, J=7.0 Hz), 1.8-2.2(m, 12H), 1.67 (s, 3H), 1.63 (s, 3H), 1.59 (s, 6H) ppm.

Mass Spec (CI-CH₄ /N₂ O,+ions) m/e 361, 359 (M+H), 137.

B. (E,E)-(5,9,13-Trimethyl-4,8,12-tetradecatrienyl)phosphinic acid,ethyl ester

A solution of 632 mg (1.75 mmol) of Part A iodide in 5 mL of diethylether was added dropwise over 25 minutes to a solution of 2.20 mL (3.74mmol) of t-butyllithium (1.7M in pentane) in 6 mL of diethyl ether underargon. After 30 minutes at -78° C. and 30 minutes at room temperature,the solution was cannulated into the addition funnel of a secondapparatus with the aid of 2 mL of diethyl ether. This solution was addedover 50 minutes to a stirred solution of 0.51 mL (3.5 mmol) of diethylchlorophosphite in 5 mL of diethyl ether at -78° C. under argon. After30 minutes at -78° C., the reaction was allowed to gradually warm to 0°C. over 50 minutes, maintained at 0° C. for one hour, and quenched with5 mL of water which had been deaerated with a stream of nitrogen. After75 minutes, the mixture was extracted with diethyl ether and the etherlayer was washed with water (four portions) and brine, dried (MgSO₄),and evaporated to afford 560 mg of a colorless oil. The oil wassubjected to flash chromatography on 70 g of SilicAR-CC7 silica gelpacked in 50:50 ethyl acetate:petroleum ether, eluted with a 60:40mixture (fractions 1-30), followed by neat ethyl acetate. Fractions19-64 gave 446.5 mg (78%) of title compound as a colorless oil. Thetitle compound may be used in forming compounds of the invention byfollowing the procedures described hereinbefore or by reacting the PartB compound with ethyl chloroformate following the procedure described inExample 15, to form Example 1, Part D product.

TLC Silica gel (ethyl acetate) R_(f) =0.22.

IR (CCl₄) 2978, 2928, 2856, 2331, 1449, 1444, 1233, 1054, 956, 804, 751cm⁻¹.

¹ H NMR (CDCl₃, 270 MHz) 6 7.07 (d, 1H, J=5.26 Hz), 5.09 (m, 3H), 4.10(m, 2H), 1.8-2.2 (m, 10H), 1.5-1.9 (s, 4H), 1.67 (s, 3H), 1.60 (s, 9H),1.36 (t, 3H, J=7 Hz) ppm.

Mass Spec (CI-CH₄ /H₂ O) (+ions) m/e 653 (2M+H) (-ions) m/e 352 (M-H).

EXAMPLE 12B (E,E)-(5,9,13-Trimethyl-4,8,12-tetradecatrienyl)-phosphinicacid, potassium salt

To a stirred solution of 446 mg (1.37 mmol) of Example 12A, Part Bcompound in 5 mL of methanol under argon was treated with 2.8 mL of 1MKOH. After stirring for 2 hours at room temperature, the methanol wasevaporated and the residue was stirred with 40 mL of diethyl ether and 5mL of 1M HCl. The ether layer was washed with water and brine, dried(MgSO₄) and evaporated to provide 391.2 mg (92%) of the free acid as acolorless liquid. The acid was purified as the ammonium salt: a solutionof the acid in 12 mL of diethyl ether was treated in a centrifuge tubewith 8 mL of ammonia saturated diethyl ether. After cooling to 0° C. andcentrifuging, the pellet was washed twice with ice cold 2:1 diethylether:hexane. The resulting granular solid was dried under vacuum toprovide an oily wax, indicative of counterion loss during drying. Theresidue was dissolved in 5 mL of 0.5M KOH and lyophilized. The residuewas chromatographed on a 18 cm height, 2.5 cm diameter column of CHP20Peluted with water (fractions 1-20) followed by 50:50 acetonitrile:water(fractions 21-50) collecting 10-15 mL fractions. Fractions 29-35werefreeze-dried to give 350.6 mg (76%) of title compound as an off-white,dense, sticky lyophilate which was dried further at high vacuum. Thetitle compound was mildly sensitive to air oxidation to thecorresponding phosphonic acid, a trace of which was noted on TLC. Theso-formed title compound may be employed to form compounds of theinvention following the procedures described hereinbefore, as well asoutlined in Example 17.

TLC Silica gel (8:1:1 n-C₃ H₇ OH:con NH₃ :H₂ O) R_(f) =0.42. Silica gel(8:1:1 CH₂ Cl₂ :CH₃ OH:CH₃ CO₂ H) R_(f) =0.44.

IR (KBr) 3360, 2968, 2922, 2858, 2289, 1665, 1449, 1159, 1064 cm⁻¹.

¹ H NMR (CD₃ OD, 400 MHz) δ 6.97 (dt, 1H, J_(P-H) =486 Hz, JH-H=2 Hz,P-H), 5.10 (m, 3H), 1.9-2.2 (m, 10H), 1.66 (s, 3H), 1.61 (s, 6H), 1.59(s, 6H), 1.4-1.7 (m, 4H) ppm.

Mass Spec (FAB+ions) m/e 375 (M+K), 359 (M+Na), 337 (M+H).

Anal. Calc'd for C₁₇ H₃₀ KO₂ P: C, 60.68; H, 8.99; P, 9.20 Found*: C,60.70; H, 9.07; P, 8.80.

EXAMPLE 13(E,E)-[[(3,7,11-Trimethyl-2,6,10-dodecatrienyl)oxy]methyl]phosphinicacid, ethyl ester

To a stirred solution of 1.302 g (2.48 mmol) of Example 5, Part Astannane in 12 mL of dry diethyl ether at -78° C. under argon was added1.8 mL (2.88 mmol) of 1.6M n-butyllithium in hexane over 7 minutes.After 30 minutes at -78° C., 1.24 mL (8.64 mmol) ofdiethylchlorophosphite was added rapidly in one portion. The reactionwas maintained at -78° C. for one hour, followed by gradual warming toroom temperature over two hours. After stirring at room temperature for50 minutes, the reaction was quenched with 2 mL of water (deoxygenatedby nitrogen bubbling), stirred for 40 minutes and extracted with diethylether. The ether layer was washed with four portions of water, dried(MgSO₄), and evaporated to provide 1.65 g of a colorless liquid. Thecrude product was dissolved in 20 mL of acetonitrile and washed withtwo-10 mL portions of hexane. The acetonitrile layer was concentrated togive 546.2 mg (67%) of title phosphonous monoester. Integration of the ¹H-NMR spectrum of the crude material suggested that it contained somefree phosphonous acid. The title compound may be employed in preparingcompounds of the invention employing procedures described hereinbefore,and was utilized in Example 15.

TLC Silica gel (Ethyl Acetate) R_(f) =0.37.

IR (CCl₄) 2970, 2926, 2917, 2857, 2380, 1667, 1448, 1383, 1191, 1108,1091, 986, 836, 781 cm⁻¹.

¹ H NMR (CDCl₃, 270 MHz) δ 7.14 (dt, 1H, J=551, 2 Hz), 5.30 (t, 1H, J=7Hz), 5.09 (br m, 2H), 4.18 (m, 2H), 4.12 (d, 2H, J=7 Hz), 3.76 (m, 2H),2.07 (m, 8H), 1.69, 1.68 (two s, 6H), 1.60 (s, 6H), 1.40 (t, 3H, J=7 Hz)ppm.

Mass Spec (CI-H₂ O/CH₄,+ions) m/e 329 (M+H).

EXAMPLE 14 (E,E)-(4,8,12-Trimethyl-3,7,11-tridecatrienyl)phosphinicacid, ethyl ester

To a stirred solution of 3.6 mL (5.76 mmol) of 1.6M t-butyllithium inpentane in 8 mL of diethyl ether at -78° C. under argon was added 995 mg(2.87 mmol) of Example 11, Part C iodide in 4 mL of diethyl ether over12 minutes to give a thin yellow suspension. After 50 minutes, thereaction was allowed to warm to 0° C. for 50 minutes. The anion solutionwas cannulated into the addition funnel of a second apparatus and wasadded dropwise over 50 minutes to 0.83 mL (5.73 mmol) of diethylchlorophosphite in 8 mL of diethyl ether at -78° C. The resulting thinsuspension was stirred for 1 hour at -78° C., allowed to warm to 0° C.over 1 hour, and then maintained at 0° C. for 1 hour. The suspension wasfiltered through dry Celite in a Schlenck filter under argon, and thesolids were washed 2×15 mL of diethyl ether. The ether was distilled offunder argon and the residue was treated with 10 mL of water containingthree drops of concentrated HCl which had been deaerated by nitrogenbubbling. The mixture was stirred for 45 minutes at room temperature,some ethyl acetate was added and stirring was continued for 30 minutes.The mixture was extracted with ethyl acetate, washed with water (5times), dried (MgSO₄) and evaporated to provide 842 mg of a colorlessoil. The crude product was flash chromatographed on 70 g of silica geleluted with 50:50 ethyl acetate:petroleum ether to provide 268.4 mg(30%) of a colorless oil which is useful in preparing compounds of theinvention.

TLC Silica gel (75:25 ethyl acetate:hexane) R_(f) =0.28.

EXAMPLE 15(E,E)-[Ethoxy[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]methyl]phosphinyl]formicacid, ethyl ester

To a stirred solution of 110.4 mg (0.336 mmol) of Example 13 ester in 2mL of diethyl ether was added 64 μL (0.504 mmol) ofchlorotrimethylsilane, 64 μL (0.672 mmol) of ethyl chloroformate and 70μL (0.504 mmol) of triethylamine. The reaction was allowed to stir for 5hours at room temperature, diluted with diethyl ether, washed with 1MHCl, water and brine, dried (MgSO₄) and evaporated to provide 117 mg oftitle ester as a colorless oil, contaminated with the correspondingphosphinic monoacid.

TLC Silica gel (ethyl acetate) R =0.57.

EXAMPLE 16(E,E)-[[(3,7,11-Trimethyl-2,6,10-dodecatrienyl)oxy]methyl]phosphinicacid

A stirred suspension of 86.6 mg (0.26 mmol) of Example 13 ester in 2 mLof water was treated with 0.5 mL of 1M KOH under argon at roomtemperature. The oil dissolved rapidly to give a cloudy solution. After30 minutes, the mixture was extracted with ethyl acetate, the aqueouslayer diluted with a second portion of ethyl acetate and acidified with1M HCl. The second organic extract was washed with water and brine,dried (MgSO₄) and evaporated to provide 63.3 mg (81%) of title compoundas a colorless oil which may be used in preparing compounds of theinvention.

TLC Silica gel (8:1:1 CH₂ Cl₂ :acetic acid:CH₃ OH) R_(f) =0.34.

EXAMPLE 17(E,E)-[Hydroxy[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]methyl]phosphinyl]formicacid, disodium salt

To a stirred solution of 63.5 mg (0.19 mmol) of Example 16 compound in 1mL of CH₂ Cl₂ was added 72 μL (0.57 mmol) of chlorotrimethylsilane, 36μL (0.38 mmol) of ethyl chloroformate and 80 mL (0.57 mmol) of triethylamine. The solution was evaporated, the residue was dissolved in 2.5 mLof 1M NaOH and stirred at room temperature for 68 hours. The solutionwas lyophilized to provide title compound contaminated with the sodiumsalt of the Example 16 compound.

TLC Silica gel (7:2:1 n-C₃ H₇ OH:con NH :H₂ O) R_(f) =0.42

EXAMPLES 18 TO 91

Following procedures of Examples 1 to 17, the following additionalcompounds may be prepared in accordance with the present invention. Itwill be appreciated that the compounds listed include all stereoisomersthereof.

    __________________________________________________________________________     ##STR55##                                                                    Ex. No.                                                                              Q.sup.4             X    p    q    R.sup.3                                                                             R.sup.2                       __________________________________________________________________________            ##STR56##          O    2    0    K     K                             19.    bond                O    0    1    Na    Na                            20.    bond                O    1    0    Na    Na                                    ##STR57##          O    1    1    Na    Na                                    ##STR58##          O    1    0    K     K                                     ##STR59##          --   2    0    CH.sub.3                                                                            K                                     ##STR60##          --   0    1    Na    Na                                    ##STR61##          O    1    1    K     K                                     ##STR62##          --   3    0    Mg                                          ##STR63##          O    1    0    Mg                                          ##STR64##          --   1    1    Na    Na                                    ##STR65##          O    2    1    Na    Na                            30.                                                                                   ##STR66##          O    2    0    K     K                                     ##STR67##          --   2    0    Mg                                  32.    bond                O    2    0    Na    Na                                    ##STR68##          --   1    1    K     K                             34.    bond                O    1    1    Na    Na                            __________________________________________________________________________     ##STR69##                                                                    Ex. No.                                                                             R.sup.7                                                                              R.sup.6                                                                             R.sup.8 p     X    q    R.sup.2                                                                            R.sup.3                       __________________________________________________________________________    35.   H      F     H       1     0    0    K    K                             36.   H      H     F       0     0    1    Na   Na                            37.   H      CH.sub.3                                                                            CH.sub.3                                                                               1    0    0    Mg                                 38.   CH.sub.3 S                                                                           CH.sub.3                                                                            H       1     0    1    K    K                             39.   F      CH.sub.3                                                                            H       2     --   0    Na   Na                            40.   CH.sub.3                                                                             CH.sub.3                                                                            H       1     0    0    K    K                             41.   H      CH.sub.3                                                                            CH.sub.3                                                                              2     --   0    Na   Na                            42.   H      CH.sub.3                                                                            F       1     0    0    K    K                             43.   H      CF.sub.3                                                                            H       1     0    0    K    K                             44.   H      F     H       0     0    1    K    K                             45.   H      CH.sub.3                                                                            (CH.sub.3).sub.3 Si                                                                   1     0    0    Na   Na                            46.   H      CH.sub.3                                                                            F       1     0    0    K    K                             __________________________________________________________________________     ##STR70##                                                                    Ex. No.                                                                             R.sup.10   R.sup.11                                                                             p     X    q     R.sup.2                                                                            R.sup.3                         __________________________________________________________________________    47.   C.sub.2 H.sub. 5                                                                         CH.sub.3                                                                             1     0    0     CH.sub.3                                                                           K                               48.   CH.sub.3   C.sub.2 H.sub.5                                                                      1     0    0     Na   Na                              49.   n-C.sub.3 H.sub.7                                                                        CH.sub.3                                                                             0     0    1     Mg                                   50.   CH.sub.3   n-C.sub.3 H.sub.7                                                                    2     --   0     K    K                               51.   CH.sub.3   n-C.sub.4 H.sub.9                                                                    1     0    0     K    CH.sub.3                        52.   t-C.sub.4 H.sub.9                                                                        CH.sub.3                                                                             1     0    0     K    K                               53.   (CH.sub.2).sub.5  2     0    1     K    K                               54.   H          H      2     --   0     Na   Na                              55.   F          F      2     --   0     Mg                                   56.   F          F      1     0    0     K    K                               57.   CH.sub.2 F CH.sub.3                                                                             1     0    0     Na   Na                              58.   CHCH.sub.2 H      0     0    1     K    K                               __________________________________________________________________________     ##STR71##                                                                    Ex. No.                                                                             R.sup.11                                                                            R.sup.6                                                                             R.sup.6 '                                                                           p     X     q     R.sup.2                                                                             R.sup.3                       __________________________________________________________________________    59.   C.sub.2 H.sub.5                                                                     C.sub.2 H.sub.5                                                                     CH.sub.3                                                                            1     0     0     K     K                             60.   CH.sub.3                                                                            CH.sub.3                                                                            C.sub.2 H.sub.5                                                                     1     0     0     Na    Na                            61.   CH.sub.3                                                                            C.sub.2 H.sub.5                                                                     C.sub.2 H.sub.5                                                                     2     --    0     Mg                                  62.   C.sub.2 H.sub.5                                                                     C.sub.2 H.sub.5                                                                     C.sub.2 H.sub.5                                                                     0     0     1     CH.sub.3                                                                            K                             63.   CH.sub.3                                                                            C.sub.2 H.sub.5                                                                     CH.sub.3                                                                            1     --    1     K     K                             64.   CH.sub.3                                                                            H     CH.sub.3                                                                            1     0     0     Na    Na                            65.   CH.sub.3                                                                            CH.sub.3                                                                            H     2     0     0     K     K                             66.   H     H     H     1     0     0     K     K                             __________________________________________________________________________    Ex.                                                                           No.                                                                           __________________________________________________________________________        ##STR72##                                                                     ##STR73##                                                                     ##STR74##                                                                 70.                                                                               ##STR75##                                                                     ##STR76##                                                                     ##STR77##                                                                     ##STR78##                                                                     ##STR79##                                                                     ##STR80##                                                                     ##STR81##                                                                     ##STR82##                                                                     ##STR83##                                                                     ##STR84##                                                                 80.                                                                               ##STR85##                                                                     ##STR86##                                                                     ##STR87##                                                                     ##STR88##                                                                     ##STR89##                                                                     ##STR90##                                                                     ##STR91##                                                                     ##STR92##                                                                     ##STR93##                                                                     ##STR94##                                                                 90.                                                                               ##STR95##                                                                     ##STR96##                                                                 __________________________________________________________________________

What is claimed is:
 1. A process for preparing a compound having theformula ##STR97## wherein R² is an alkali metal or lower alkyl; R³ is analkali metal or lower alkyl; R is R¹ --(CH₂)_(n) -- or R¹ --(CH₂)_(m)OCH₂ --, wherein n is an integer from 1 to 4 and m is an integer from 0to 3; and R¹ is R⁵ --Q¹ --Q² --Q³ -- wherein Q¹, Q² and Q³ areindependently: ##STR98## or a bond with the stipulation that if Q¹ is abond, then Q² and Q³ must be bonds, and if Q² is a bond, then Q³ is abond; if any of Q¹, Q² and Q³ is ##STR99## then R⁶ is H, lower alkyl,fluoro or fluoroalkyl; R⁷ is H, fluoro, lower alkyl or alkythio; R⁸ isH, fluoro, trimethylsilyl or lower alkyl; R⁹ is H, or lower alkyl;##STR100## R¹⁶ --C.tbd.C--CH₂ -- (wherein R¹⁶ is lower alkyl or H), orCH₃ (CH₂)_(p) -- where p is 2 to 7; R¹⁰ and R¹¹ are independentlyhydrogen, lower alkyl, fluoro, lower alkenyl or fluoroalkyl or R¹⁰ andR¹¹ can be taken together to form (CH₂)_(s), where s is 2 to 7; R¹² ishydrogen, lower alkyl, fluoro or lower alkenyl; R¹³ and R¹⁴ areindependently lower alkyl; with the proviso that if all of Q¹, Q² and Q³are bonds, then R¹⁰ and R¹¹ cannot both be H, and R⁵ cannot be CH₃(CH₂)_(p) --, with p≦4, including all stereoisomers thereof, whichcomprises treating a compound of the structure

    R-Metal

where R is as defined above, with a phosphite of the structure

    ClP(OR.sup.2).sub.2

wherein R² is lower alkyl, to form a phosphonite of the structure

    R--P(OR.sup.2).sub.2

and treating the phosphonite with an alkylating agent of the structure

    ClCO.sub.2 R.sup.3

to form the phosphinylformate diester of the structure ##STR101## andoptionally, if desired, treating the so-formed phosphinylformate diesterwith a dealkylating agent and one molar equivalent of alkali metal baseto form the metal salt of the structure ##STR102## and optionallytreating said metal salt with a strong acid to form the acid of thestructure ##STR103## or optionally, if desired, treating the so-formedphosphinylfomrate diester with a dealkylating agent and/or excess alkalimetal base to form the dimetal salt of the structure ##STR104##
 2. Theprocess as defined in claim 1 wherein R is R¹ --CH)₂ n--.
 3. The processas defined in claim 1 wherein R is R¹ --CH₂)_(m) --O--CH₂ --.
 4. Theprocess as defined in claim 1 wherein R¹ is ##STR105##
 5. The process asdefined in claim 1 wherein R is R¹ --CH₂)_(n) -- and n is 1, 2 or
 3. 6.The process as defined in claim 1 wherein R is R¹ --CH₂)_(m) OCH₂ --wherein m is
 1. 7. The process as defined in claim 1 wherein R² is ametal ion and/or R³ is a metal ion.